Process for typing of HCV isolates

ABSTRACT

The invention relates to a process for genotyping any HCV isolate present in a biological sample, previously identified as being HCV positive, and for classifying said isolate according to the percentage of homology with other HCV isolates, comprising the steps of:
         contacting said sample in which the ribonucleotides or deoxyribonucleotides have been made accessible, if need be, under suitable denaturation, with at least one probe from about 10 to about 40 nucleotides, with said probe being liable to hybridize to a region being in the domain extending from nucleotide at position −291 to nucleotide at position −66 of the 5′ untranslated region of one of the HCV isolates represented by their cDNA sequences, with said numbering of position beginning with the first ATG codon of the open reading frame encoding the HCV polyprotein, or with said probe being complementary to the above-defined probes,   detecting the complexes possibly formed between said probe and the nucleotide sequence of the HCV isolate to be identified.

This application is a divisional of Application No. 10/412,290, filedApr. 14, 2003 (which issued as U.S. Pat. No. 7,258,977 on Aug. 21,2007), which is a continuation of Application No. 09/899,044, filed Jul.6, 2001 (which issued as U.S. Pat. No. 6,548,244), which is a divisionalof Application No. 09/378,900, filed Aug. 23, 1999 (which issued as U.S.Pat. No. 6,495,670), which is a divisional of Application No.09/044,665, filed Mar. 19, 1998 (which issued as U.S. Pat. No.6,051,696), which is a divisional of Application No. 08/256,568, filedJul. 18, 1994 , which issued as U.S. Pat. No. 5,846,704, which is a 371U.S. National Phase of PCT/EP93/03325, filed Nov. 26, 1993, which claimsbenefit of EP 92 403 222.0, filed Nov. 27, 1992 and EP 93 402 129.6,filed Aug. 31, 1993, the entire content of each of which is herebyincorporated by reference in this application

The invention relates to the use of probes targeting sequences from the5′ untranslated region of HCV for genotyping of HCV isolates.

The invention also relates to a process for genotyping of HCV isolates.

The invention also relates to a kit for genotyping of HCV isolates.

Hepatitis C viruses (HCV) are a family of positive-stranded, envelopedRNA viruses causing the majority of non-A, non-B (NANB) hepatitis: Theirgenomic organization indicates a close relationship to the Pestiviridaeand Flaviviridae. The sequences of cDNA clones covering the completegenome of several prototype isolates have already been completelydetermined (Kato et al., 1990; Choo et al., 1991; Okamoto et al., 1991;Takamizawa et al., 1991; Okamoto et al., 1992b). These genomes are about9500 base pairs long. The isolates reported by Kato, Takamizawa, andChoo contain an open reading frame (ORF) of 3010 or 3011 amino acids,and those reported by Okamoto encode 3033 amino acids. Comparison ofthese isolates shows a considerable variability in the envelope (E) andnon-structural (NS) regions, while the 5′ untranslated region (UR) and,to a lesser extent, the core region are highly conserved.

Using cloned sequences of the NS3 region, Kubo et al. (1989) compared aJapanese and an American isolate and found nearly 80% nucleotide and 92%amino acid homology. The existence of sequence variability was furtherdocumented when sequences of the 5′ UR, core, and E1 regions becameavailable (HC-J1 and HC-J4; Okamoto et al., 1990). After the isolationof several NS5 fragments in Japanese laboratories, two groups, K1 andK2, were described (Enomoto et al., 1990). A comparison of the“American-like” isolate PT-1 with K1, which was more prevalent in Japan,showed that they represent closely related but different subtypes withan intergroup nucleotide identity of about 80%. The K2 sequence was moredistantly related to both K1 and PT-1, because homologies of only 67% atthe nucleic acid level, and 72% at the amino acid level were observed.Moreover, K2 could be divided into two groups, K2a and K2b, also showingintergroup nucleotide homologies of about 80%. Nucleotide sequenceanalysis in the 5′ UR showed 99% identity between K1 and PT-1, and atmost 94% identity between K1 and K2, enabling the use of the 5′ UR forrestriction fragment length polymorphism (RFLP) and classification ofHCV into groups K1 and K2 (Nakao et al., 1991). Further evidence for asecond group was given by the complete sequence of HC-J6 and HC-J8, twosequences related to the K2 group (Okamoto et al., 1991; Okamoto et al.,1992b). A phylogenetic tree of HCV containing four branches (i.e., TypeI: HCV-1 and HCV-H; Type HCV-J, -BK, HC-J4; Type III: HC-J6; Type IV:HC-J8) was proposed by Okamoto et al. (1992b). However, nucleic acidsequence homologies of 79% can be observed between Type I and Type II,and also between Type III and IV. A lesser degree of relatedness betweenthe first group (Type I and II) and the second group (Types III and IV)of only 67-68% exists. Moreover, a new type of HCV, HCV-T, was detectedin Thailand after studying NS5 regions (Mori et al., 1992). HCV-T had asequence homology of about 65% with all other known NS5 sequences, andtwo groups could be detected, HCV-Ta and HCV-Tb, which again exhibitednucleic acid sequence homologies of about 80%. Elucidation of thephylogenetic relationship of a similar new group found in Britishisolates with Type I to IV was possible by analyzing the conserved partsof the 5′ UR, core, NS3, and NS5 regions (Chan et al., 1992a). A newphylogenetic tree was proposed, whereby ‘type 1’ corresponds with Type Iand II, ‘type 2’ with Type III and IV, and ‘type 3’ with their ownisolates E-b1 to E-b8 and HCV-T. Some sequences of the 5′ UR of isolatesfrom ‘type 3’ were also reported by others (Bukh et al., 1992; Cha etal., 1992; Lee et al., 1992).

Several patent applications have addressed the problem of detecting thepresence of HCV by means of probes derived from the genome of type 1 HCVisolates (WO 92/02642, EP 419 182, EP 398 748, EP 469 438 and EP 461863). Furthermore, the 5′ UR of HCV isolates has been proven to be agood candidate for designing probes and primers for general HCVdetection (Cha et al., 1991; Inchaupse et al., 1991). However, none ofthese patent applications presents a method for identifying the typeand/or subtype of HCV present in the sample to be analyzed.

The demonstration that different HCV genotype infections resulted indifferent serological reactivities (Chan et al., 1991) and responses tointerferon IFN-γ treatment (Pozatto et al., 1991; Kanai et al., 1992;Yoshioka et al., 1992) stresses the importance of HCV genotyping. Untilnow, this could only be achieved by large sequencing efforts in thecoding region or in the 5′ UR, or by polymerase chain reactions (PCR) onHCV cDNA with type-specific sets of core primers (Okamoto et al. 1992a),or by (RFLP) analysis in the 5′ UR or in the NS5 region (Nakao et al.,1991; Chan et al., 1992b). However, none of these above-mentioned patentapplications or publications offers a reliable method for identifyingthe type or subtype of HCV present in the sample to be analyzed,especially since typing is laborious and subtyping seems to be even morelaborious or impossible by means of these methods. In this respect, itcan be noted that Lee et al. (1992) attempt to distinguish between theHCV isolates HCV 324 and HCV 324X by means of PCR fragments from the 5′UR of the genomes of these isolates. The results demonstrate that these5′ UR probes do not show a specific reactivity with the genome of therespective isolate from which they were derived.

Consequently, the aim of the present invention is to provide a methodfor the rapid and indisputable determination of the presence of one orseveral genotypes of HCV present in a biological sample and indisputablyclassifying the determined isolate(s).

Another aim of the invention is to provide a process for identifying yetunknown HCV types or subtypes.

Another aim of the invention is to provide a process enabling theclassification of infected biological fluids into different serologicalgroups unambiguiously linked to types and subtypes at the genomic level.

Another aim of the invention is to provide a kit for rapid detection ofthe presence or absence of different types or subtypes of HCV.

The invention relates to the use of at least one probe, with said probebeing (i) capable of hybridizing to a genotype specific target region,present in an analyte strand, in the domain extending from thenucleotides at positions −291 to −66 of the 5′ untranslated region (UR)of one of the HCV isolates, or with said probe being (ii) complementaryto any of the above-defined probes, for genotyping HCV isolates presentin a biological sample.

The invention relates to the use of at least one probe preferablycontaining from about 5 to about 50 nucleotides, more preferably fromabout 10 to about 40 nucleotides, and most preferably containing fromabout 15 to about 30 nucleotides, with said probe being (i) capable ofliable to hybridizing to a genotype specific target region present in ananalyte strand in the domain extending from the nucleotides at positions−291 to −66 of the 5′ UR of one of the HCV isolates represented by theircDNA sequences, for example represented by their cDNA sequences in FIG.2, with said negative numbering of the nucleotide positions starting atthe nucleotide preceding the first ATG codon of the open reading frameencoding the HCV polyprotein, or with said probe being (ii)complementary to the above-defined probes, for (in vitro) genotyping HCVisolates present in a biological sample, with said sample being possiblypreviously identified as being HCV positive.

The above mentioned process may be used for classifying said isolateaccording to the percentage of homology with other HCV isolates,according to the fact that isolates belonging to the same type:

exhibit homology of more than 74% at the nucleic acid level in thecomplete genome;

or exhibit homology of more than 74% at the nucleic acid level in theNS5 region between nucleotide positions 7935 and 8274;

or of which the complete polyprotein shows more than 78% homology at theamino acid level;

or of which the NS5 region between amino acids at positions 2646 and2758 shows more than 80% homology at the amino acid level;

and according to the fact that HCV isolates belonging to the samesubtype exhibit homology of more than 90% at the nucleic acid level inthe complete genome and of more than 90% at the amino acid level in thecomplete polyprotein.

More preferably the above mentioned process relates to theclassification of HCV isolates according to the fact that,

(1) based on phylogenetic analysis of nucleic acid sequences in the NS5bregion between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al, 1991),isolates belonging to the same HCV type show nucleotide distances ofless than 0.34, usually less than 0.33, and more usually of less than0.32, and isolates belonging to the same subtype show nucleotidedistances of less than 0.135, usually of less than 0.13, and moreusually of less than 0.125, and consequently isolates belonging to thesame type but different subtypes show nucleotide distances ranging from0.135 to 0.34, usually ranging from 0.14 to 0.33, and more usuallyranging from 0.15 to 0.32, and isolates belonging to different HCV typesshow nucleotide distances greater than 0.34, usually greater than 0.35,and more usually of greater than 0.36,

(2) based on phylogenetic analysis of nucleic acid sequences in thecore/E1 region between nucleotides 378 and 957, isolates belonging tothe same HCV type show nucleotide distances of less than 0.38, usuallyof less than 0.37, and more usually of less than 0.36, and isolatesbelonging to the same subtype show nucleotide distances of less than0.17, usually of less than 0.16, and more usually of less than 0.15, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.15 to 0.38, usually rangingfrom 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.36, usually more than 0.365, and more usually of greaterthan 0.37,

(3) based on phylogenetic analysis of nucleic acid sequences in theNS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) orbetween nucleotides 4993 and 5621 (Kato et al., 1990) or betweennucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging tothe same HCV type show nucleotide distances of less than 0.35, usuallyof less than 0.34, and more usually of less than 0.33, and isolatesbelonging to the same subtype show nucleotide distances of less than0.19, usually of less than 0.18, and more usually of less than 0.17, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.17 to 0.35, usually rangingfrom 0.18 to 0.34, and more usually ranging from 0.19 to 0.33, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.33, usually greater than 0.34, and more usually ofgreater than 0.35.

The term “genotyping” refers to either typing and/or subtyping. A methodfor ‘genotyping’ HCV isolates is considered to, at least partly,classify HCV isolates into genotypes. A HCV ‘genotype’ is a group of HCVisolates with related sequences. Said related sequences are defined asshowing nucleotide distances as indicated above and as illustrated inexample 9. Both larger groups (HCV types) and smaller groups (HCVsubtypes) have been shown to be related. A HCV type always includes oneor more HCV subtypes. Consequently, a method for genotyping can aim attyping (classification into HCV types) of HCV isolates without the needfor subtyping (classification into HCV subtypes), or, in a preferredembodiment, subtyping can be aimed at. It should be understood thatclassification into subtypes inherently yields data for classificationinto types.

The expression “genotype specific target region” refers at least onenucleotide variation observed between different HCV genotypes in the 5′untranslated region (UR) as can be readily deduced from FIGS. 2 and 4.

The term “HCV polyprotein” refers to the HCV polyprotein of the HCV-Jisolate (Kato et al., 1990), which belongs to subtype 1b.

The expression “probe” corresponds to any polynucleotide which forms ahybrid with a target sequence present in a certain HCV isolate on thebasis of complementarity. Such a probe may be composed of DNA, RNA, orsynthetic nucleotide analogs. The probes of the invention can beincubated with an analyte strand immobilized to a solid substrate. In apreferred embodiment of the invention, the probes themselves can beimmobilized to a solid substrate. These probes may further includecapture probes, characterized as being coupled to a binding moleculewhich in turn is directly or indirectly bound to a solid substrate, ormay also include label probes, characterized in that they carry adetectable label.

The invention relates to a process for genotyping HCV isolates presentin a biological sample; comprising the steps of:

-   -   contacting said sample in which the ribonucleotides or        deoxyribonucleotides have been made accessible, if need be,        under suitable denaturation, with at least one probe, with said        probe being (i) capable of hybridizing to a region in the domain        extending from nucleotides at positions −291 to −66 of the 5′        untranslated region of one of the HCV isolates, or with said        probe being (ii) complementary to any of the above-defined        probes, and,    -   detecting the complexes possibly formed between said probe and        the nucleotide sequence of the HCV isolate to be identified.

The invention relates also to a process for genotyping an HCV isolatepresent in a biological sample, comprising the steps of:

-   contacting said sample in which the ribonucleotides and    deoxyribonucleotides have been made accessible, if need be, under    suitable denaturation, with at least one probe from preferably from    about 5 to 50, more preferably from about 10 to about 40 nucleotides    most preferably from about 15 to about 30 nucleotides, with said    probe being (i) capable of hybridizing to a region in the domain    extending from nucleotides at positions −291 to −66 of the 5′ UR of    one of the HCV isolates represented by their cDNA sequences, for    example represented by their cDNA sequences in FIG. 2, with said    negative numbering of position starting at the nucleotide preceding    the first ATG codon of the open reading frame encoding the HCV    polyprotein, or with said probe being complementary to the    above-defined probes,-   detecting the complexes possibly formed between said probe and the    nucleotide sequence of the HCV isolate to be identified, and,    inferring the type(s) of HCV isolates present from the hybridization    pattern.

The above mentioned method can be considered as a method for classifyingsaid isolate according to the percentage of homology with other HCVisolates, according to the fact that isolates belonging to the sametype:

-   exhibit homology of more than 74% at the nucleic acid level in the    complete genome,-   or exhibit homology of more than 74% at the nucleic acid level in    the NS5 region between nucleotide positions 7935 and 8274,-   or of which the complete polyprotein shows more than 78% homology at    the amino acid level,-   or of which the NS5 region between amino acids at positions 2646 and    2758 shows more than 80% homology at the amino acid level,-   and according to the fact that HCV isolates belonging to the same    subtype exhibit homology of more than 90% at the nucleic acid level    in the complete genome and of more than 90% at the amino acid level    in the complete polyprotein.

More preferably, said method relates to the classification of HCVisolates according to the fact that,

(1) based on phylogenetic analysis of nucleic acid sequences in the NS5bregion between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al, 1991),isolates belonging to the same HCV type show nucleotide distances ofless than 0.34, usually less than 0.33, and more usually of less than0.32, and isolates belonging to the same subtype show nucleotidedistances of less than 0.135, usually of less than 0.13, and moreusually of less than 0.125, and consequently isolates belonging to thesame type but different subtypes show nucleotide distances ranging from0.135 to 0.34, usually ranging from 0.14 to 0.33, and more usuallyranging from 0.15 to 0.32, and isolates belonging to different HCV typesshow nucleotide distances greater than 0.34, usually greater than 0.35,and more usually of greater than 0.36,

(2) based on phylogenetic analysis of nucleic acid sequences in thecore/E1 region between nucleotides 378 and 957, isolates belonging tothe same HCV type show nucleotide distances of less than 0.38, usuallyof less than 0.37, and more usually of less than 0.36, and isolatesbelonging to the same subtype show nucleotide distances of less than0.17, usually of less than 0.16, and more usually of less than 0.15, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.15 to 0.38, usually rangingfrom 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.36, usually more than 0.365, and more usually of greaterthan 0.37,

(3) based on phylogenetic analysis of nucleic acid sequences in theNS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) orbetween nucleotides 4993 and 5621 (Kato et al., 1990) or betweennucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging tothe same HCV type show nucleotide distances of less than 0.35, usuallyof less than 0.34, and more usually of less than 0.33, and isolatesbelonging to the same subtype show nucleotide distances of less than0.19, usually of less than 0.18, and more usually of less than 0.17, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.17 to 0.35, usually rangingfrom 0.18 to 0.34, and more usually ranging from 0.19 to 0.33, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.33, usually greater than 0.34, and more usually ofgreater than 0.35.

The term “analyte strand” corresponds to a single- or double-strandednucleic acid molecule which is suspected to contain sequences which maybe present in a biological sample, with said analyte strand beingdirectly detected or detected after amplification. This analyte strandis preferentially positive- or negative-stranded RNA, cDNA, or amplifiedcDNA.

The expression “biological sample” may refer to any biological sample(tissue or fluid) containing HCV sequences and refers more particularlyto blood serum or plasma samples.

The detection of hybrids formed between the type- or subtype-specifictarget region, if present, and the probes as mentioned above depends onthe nature of the reporter molecule used (either present on the probe oron the analyte strand to be targeted) and may be determined by means ofcolorimetric, fluorescent, radiometric detection or any other methodcomprised in the state of the art.

The term “(HCV) isolates” refers to any biological fluid containinghepatitis C virus genetic material obtained from naturally infectedhumans or experimentally infected animals, and also refers to fluidscontaining hepatitis C virus genetic material which has been obtainedfrom in vitro experiments. For instance, from in vitro cultivationexperiments, both cells and growth medium can be employed as a source ofHCV genomes material.

The expression “hybridize” or “target” refers to a hybridizationexperiment carried out according to any method known in the art, andallowing the detection of homologous targets (including one or fewmismatches) or preferably completely homologous targets (no mismatchesallowed).

In the present invention, a sensitive PCR protocol has been used for thehighly conserved 5′ UR with sets of nested, universal primers. Positionsand sequences of these primers were derived from the sequences ofpreviously reported type 1 and 2 sequences, and the type 3 sequence BR56(FIG. 2). The obtained amplification product was hybridized tooligonucleotides directed against the variable regions of the 5′ UR,immobilized as parallel lines on membrane strips (reverse-hybridizationprinciple). This hybridization assay, called line probe assay (LiPA), isa rapid assay, by means of which previously poorly described isolatessimilar to Z4, Z6, and Z7 (Bukh et al., 1992) were detected. A new type4 classification is proposed for these strains of HCV. Other isolatessimilar to BE95 and BE96, and to SA1 (Cha et al., 1992) can bedistinguished and it is proposed to classify such isolates as type 5a.Isolates similar to HK2 (Bukh et al., 1992) can be distinguished and anew type 6a classification is proposed. A new genotype was detected inisolate BE98, and it is proposed to classify this isolate into HCV type3, subtype 3c. Another new sequence was detected in GB438, which couldbe classified as 4f. This LiPA technology allows an easy and fastdetermination of HCV types and their subtypes present in patient serum.

According to a preferred embodiment of the invention, a set of probescomprising at least two probes is used.

According to a preferred embodiment, in the process of the invention theprobe used targets a region of at least 5 nucleotides in one of thefollowing domains:

-   -   a) the one extending from nucleotide at position −293 to        nucleotide at position −278 in FIG. 2,    -   b) the one extending from nucleotide at position −275 to        nucleotide at position −260 in FIG. 2,    -   c) the one extending from nucleotide at position −253 to        nucleotide at position −238 in FIG. 2,    -   d) the one extending from nucleotide at position −244 to        nucleotide at position −229 in FIG. 2,    -   e) the on extending from nucleotide at position −238 to        nucleotide at position −223 in FIG. 2,    -   f) the one extending from nucleotide at position −170 to        nucleotide at position −155 in FIG. 2,    -   g) the one extending from nucleotide at position −141 to        nucleotide at position −117 in FIG. 2,    -   h) the one extending from nucleotide at position −83 to        nucleotide at position −68 in FIG. 2,    -   i) the one extending from nucleotide at position −103 to        nucleotide at position −88 in FIG. 2,    -   j) the one extending from nucleotide at position −146 to        nucleotide at position −130.

Regions −170 to −155 and −141 to −117 represent variable regions in thelinear sequence which may be part of the same stem in the viral RNA.Consequently mutations in one region may be complemented by anothermutation in another region to allow or disallow RNA duplex formation.Variation is expected to occur at the same positions in other new typesof HCV as well and, therefore, these variable regions might remaininstrumental for the discrimination between all current and yet-to-bediscovered types of HCV.

According to yet another embodiment the present invention relates to aprobe comprising a sequence such that it targets at least one of thefollowing sequences:

AAT TGC CAG GAC GAC C (SEQ ID NO 5) TCT CCA GGC ATT GAG C (SEQ ID NO 6)CCG CGA GAC TGC TAG C (SEQ ID NO 7) TAG CGT TGG GTT GCG A (SEQ ID NO 8)TTR CCG GRA AGA CTG G (SEQ ID NO 9) TGR CCG GGC ATA GAG T (SEQ ID NO 10)TTA CCG GGA AGA CTG G (SEQ ID NO 11) TGA CCG GAC ATA GAG T (SEQ ID NO12) AAT CGC TGG GGT GAC C (SEQ ID NO 13) TTT CTG GGT ATT GAG C (SEQ IDNO 14) TCT TGG AGC AAC CCG C (SEQ ID NO 15) TCT TGG AAC AAC CCG C (SEQID NO 16) AAT YGC CGG GAT GAC C (SEQ ID NO 17) TTC TTG GAA CTA ACC C(SEQ ID NO 18) TTT CCG GGC ATT GAG C (SEQ ID NO 19) TTG GGC GYG CCC CCGC (SEQ ID NO 20) CCG CGA GAT CAC TAG C (SEQ ID NO 21) CCG GGA AGA CTGGGT C (SEQ ID NO 22) CCG GAA AGA CTG GGT C (SEQ ID NO 23) ACC CAC TCTATG CCC G (SEQ ID NO 24) ACC CAC TCT ATG TCC G (SEQ ID NO 25) ATA GAGTGG GTT TAT C (SEQ ID NO 26) TCT GCG GAA CCG GTG A (SEQ ID NO 27) AATTGC CAG GAY GAC C (SEQ ID NO 28) GCT CAG TGC CTG GAG A (SEQ ID NO 29)CCG CGA GAC YGC TAG C (SEQ ID NO 30) CCC CGC AAG ACT GCT A (SEQ ID NO31) CGT ACA GCC TCC AGG C (SEQ ID NO 32) GGA CCC AGT CTT CCT G (SEQ IDNO 33) TGC CTG GTC ATT TGG G (SEQ ID NO 34) TKT CTG GGT ATT GAG C (SEQID NO 35) CCG CAA GAT CAC TAG C (SEQ ID NO 36) GAG TGT TGT ACA GCC T(SEQ ID NO 37) AAT CGC CGG GAT GAC C (SEQ ID NO 38) GAG TGT TGT GCA GCCT (SEQ ID NO 39) AAT CGC CGG GAC GAC C (SEQ ID NO 40) AAT GCC CGG CAATTT G (SEQ ID NO 41) AAT CGC CGA GAT GAC C (SEQ ID NO 42) AAT GCT CGGAAA TTT G (SEQ ID NO 43) GAG TGT CGA ACA GCC T (SEQ ID NO 44) AAT TGCCGG GAT GAC C (SEQ ID NO 45) TCT CCG GGC ATT GAG C (SEQ ID NO 46) AATTGC CGG GAC GAC C (SEQ ID NO 47) GGG TCC TTT CCA TTG G (SEQ ID NO 48)AAT CGC CAG GAT GAC C (SEQ ID NO 49) TGC CTG GAA ATT TGG G (SEQ ID NO50) GAG TGT CGT ACA GCC T (SEQ ID NO 51) AGT YCA CCG GAA TCG C (SEQ IDNO 52) GGA ATC GCC AGG ACG A (SEQ ID NO 53) GA TCG CCG GGT TGA C (SEQ IDNO 54) GAG TGT TGT ACA GCC TCC (SEQ ID NO 93) TGC CCG GAA ATT TGG GC(SEQ ID NO 94) TGC CCG GAG ATT TGG G (SEQ ID NO 95) GAG TGT CGA ACA GCCTC (SEQ ID NO 96)

wherein Y represents T or C

K represents G or T

and R represents G or A

-   -   or the corresponding sequence wherein T has been replaced by U,    -   or the sequences which are complementary to the above-defined        sequences.

According to another advantageous embodiment of the invention, at leasttwo of the above-mentioned probes or a mixture of two of these probes isused to discriminate between various HCV types or subtypes as definedbelow.

According to a preferred embodiment of the process of the invention, foreach type or subtype of HCV to be determined, a set of two differentprobes or a mixture of two different probes is used, with each probe ofthe set or of the mixture respectively targeting a different regionchosen among the regions as defined above, and more particularly whereinthe two probes, in said set or in said mixture, consist of 10 to 40contiguous nucleotides respectively targeting two regions respectivelychosen from among the following pairs of domains:

-   -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −141 to nucleotide at position −117 in FIG. 2,    -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −103 to nucleotide at position −88 in FIG. 2,    -   the one extending from nucleotide at position −141 to nucleotide        at position −117 in FIG. 2 and the one extending from nucleotide        at position −103 to nucleotide at position −88 in FIG. 2,    -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −83 to nucleotide at position −68 in FIG. 2,    -   the one extending from nucleotide at position −141 to nucleotide        at position −117 in FIG. 2 and the one extending from nucleotide        at position −83 to nucleotide at position −68 in FIG. 2,    -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −146 to nucleotide at position −130 in FIG. 2,    -   the one extending from nucleotide at position −132 to nucleotide        at position −117 in FIG. 2 and the one extending from nucleotide        at position −146 to nucleotide at position −130 in FIG. 2,    -   the one extending from nucleotide at position −146 to nucleotide        at position −130 in FIG. 2 and the one extending from nucleotide        at position −103 to nucleotide at position −88 in FIG. 2.

The invention also relates to a probe having a sequence such that ittargets:

-   -   the following sequence: TTC TTG GAA CTA ACC C,    -   or the corresponding sequence wherein T has been replaced by U,    -   or the sequences which are complementary to the above-defined        sequences.

The invention also relates to a set of two probes or mixtures of twoprobes wherein each of the two probes consists of 10 to 40 contiguousnucleotides, and wherein the two probes respectively target two regionsrespectively chosen from among the following pairs of domains:

-   -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −141 to nucleotide at position −117 in FIG. 2,    -   one extending from nucleotide at position −170 to nucleotide at        position −155 in FIG. 2 and the one extending from nucleotide at        position −103 to nucleotide at position −88 in FIG. 2,    -   the one extending from nucleotide at position −141 to nucleotide        at position −117 in FIG. 2 and the one extending from nucleotide        at position −103 to nucleotide at position −88 in FIG. 2,    -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −83 to nucleotide at position −68 in FIG. 2,    -   the one extending from nucleotide at position −141 to nucleotide        at position −117 in FIG. 2 and the one extending from nucleotide        at position −83 to nucleotide at position −68 in FIG. 2,    -   the one extending from nucleotide at position −170 to nucleotide        at position −155 in FIG. 2 and the one extending from nucleotide        at position −146 to nucleotide at position −130 in FIG. 2,    -   the one extending from nucleotide at position −132 to nucleotide        at position −117 in FIG. 2 and the one extending from nucleotide        at position −146 to nucleotide at position −130 in FIG. 2,    -   the one extending from nucleotide at position −146 to nucleotide        at position −130 in FIG. 2 and the one extending from nucleotide        at position −103 to nucleotide at position −88 in FIG. 2.

According to a preferred embodiment, the invention relates to a processfor typing HCV isolates as belonging to at least one of the followingHCV types: HCV type 1, HCV type 2, HCV type 3, HCV type 4, HCV type 5,HCV type 6 from a biological sample liable to contain it, and comprisesthe steps of:

-   -   contacting said sample in which the ribonucleotides or        deoxyribonucleotides have been made accessible, if need be under        suitable denaturation, with at least one probe being capable of        hybridizing to a region in the domain extending from nucleotide        at position −291 to nucleotide at position −66 of the 5′ UR of        HCV isolates represented by their cDNA sequences in FIGS. 2 and        4, with said negative numbering of the nucleotide position        starting at the nucleotide preceding the first ATG codon in the        open reading frame encoding the HCV polyprotein or with said        probe being complementary to the above-defined probes;    -   detecting the complexes possibly formed between said probe and        the target region, and,    -   inferring the HCV types present from the observed hybridization        pattern.

According to a preferred embodiment, the invention relates to a processfor typing HCV isolates as belonging to at least one of the followingHCV types: HCV type 1, HCV type 2, HCV type 3, HCV type 4, HCV type 5,and HCV type 6, and is such that the probes used are able to target oneof the following target regions or said regions wherein T has beenreplaced by U, or the regions which are complementary to the above-saidregions:

for HCV type 1 and 6: AAT TGC CAG GAC GAC C (No. 5) TCT CCA GGC ATT GAGC (No. 6) AAT TGC CAG GAY GAC C (No. 28) for HCV type 1: GCT CAG TGC CTGGAG A (No. 29) for HCV type 2: TAG CGT TGG GTT GCG A (No. 8) TTR CCG GRAAGA CTG G (No. 9) TGR CCG GGC ATA GAG T (No. 10) TTA CCG GGA AGA CTG G(No. 11) TGA CCG GAC ATA GAG T (No. 12) CGT ACA GCC TCC AGG C (No. 32)CCG GGA AGA CTG GGT C (No. 22) CCG GAA AGA CTG GGT C (No. 23) ACC CACTCT ATG CCC G (No. 24) ACC CAC TCT ATG TCC G (No. 25) ATA GAG TGG GTTTAT C (No. 26) GGA CCC AGT CTT CCT G (No. 33) TGC CTG GTC ATT TGG G (No.34) for HCV type 3: AAT CGC TGG GGT GAC C (No. 13) TTT CTG GGT ATT GAG C(No. 14) CCG CGA GAT CAC TAG C (No. 21) CCG CAA GAT CAC TAG C (No. 36)GAA TCG CCG GGT TGA C (No. 54) for HCV type 4 and 5: AAT YGC CGG GAT GACC (No. 17) for HCV type 4: TTC TTG GAA CTA ACC C (No. 18) for HCV type4, 3c TTT CCG GGC ATT GAG C (No. 19) and 3b: for HCV type 4 and 3b: AATCGC CGG GAT GAC C (No. 38) for HCV type 4: GAG TGT TGT ACA GCC T (No.37) GAG TGT TGT GCA GCC T (No. 39) AAT CGC CGG GAC GAC C (No. 40) AATGCC CGG CAA TTT G (No. 41) AAT CGC CGA GAT GAC C (No. 42) AAT GCT CGGAAA TTT G (No. 43) AAT CGC CAG GAT GAC C (No. 49) TGC CTG GAA ATT TGG G(No. 50) GGA ATC GCC AGG ACG A (No. 53) for HCV type 5: AAT TGC CGG GATGAC C (No. 45) AAT TGC CGG GAC GAC C (No. 47) TCT CCG GGC ATT GAG C (No.46) GAG TGT CGA ACA GCC T (No. 44) for HCV type 6: GGG TCC TTT CCA TTG G(No. 48)wherein Y represents C or T, and K represents G or T, or the probes usedare a set of two probes chosen from among the above-defined probes.

The invention also relates to the use of the above-defined method fordetermining the type(s) of HCV isolates present in a biological sample.

The term “type” corresponds to a group of HCV isolates of which thecomplete genome shows more than 74% homology at the nucleic acid level,or of which the NS5 region between nucleotide positions 7935 and 8274shows more than 74% homology at the nucleic acid level, or of which thecomplete HCV polyprotein shows more than 78% homology at the amino acidlevel, or of which the NS5 region between amino acids at positions 2646and 2758 shows more than 80% homology at the amino acid level, togenomes of the other isolates of the group, with said numberingbeginning with the first ATG codon or methionine of the HCV polyproteinof the HCV-1 isolate (Kato et al., 1990). Isolates belonging todifferent types of HCV exhibit homologies of less than 74% at thenucleic acid level and less than 78% at the amino acid level. Isolatesbelonging to the same type usually show homologies of about 92 to 95% atthe nucleic acid level and 95 to 96% at the amino acid level whenbelonging to the same subtype, and those belonging to the same type butdifferent subtypes preferably show homologies of about 79% at thenucleic acid level and 85-86% at the amino acid level. More preferably,classification of HCV isolates should be performed according to the factthat,

(1) based on phylogenetic analysis of nucleic acid sequences in the NS5bregion between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al, 1991),isolates belonging to the same HCV type show nucleotide distances ofless than 0.34, usually less than 0.33, and more usually of less than0.32, and isolates belonging to the same subtype show nucleotidedistances of less than 0.135, usually of less than 0.13, and moreusually of less than 0.125, and consequently isolates belonging to thesame type but different subtypes show nucleotide distances ranging from0.135 to 0.34, usually ranging from 0.14 to 0.33, and more usuallyranging from 0.15 to 0.32, and isolates belonging to different HCV typesshow nucleotide distances greater than 0.34, usually greater than 0.35,and more usually of greater than 0.36,

(2) based on phylogenetic analysis of nucleic acid sequences in thecore/E1 region between nucleotides 378 and 957, isolates belonging tothe same HCV type show nucleotide distances of less than 0.38, usuallyof less than 0.37, and more usually of less than 0.36, and isolatesbelonging to the same subtype show nucleotide distances of less than0.17, usually of less than 0.16, and more usually of less than 0.15, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.15 to 0.38, usually rangingfrom 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.36, usually more than 0.365, and more usually of greaterthan 0.37,

(3) based on phylogenetic analysis of nucleic acid sequences in theNS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) orbetween nucleotides 4993 and 5621 (Kato et al., 1990) or betweennucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging tothe same HCV type show nucleotide distances of less than 0.35, usuallyof less than 0.34, and more usually of less than 0.33, and isolatesbelonging to the same subtype show nucleotide distances of less than0.19, usually of less than 0.18, and more usually of less than 0.17, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.17 to 0.35, usually rangingfrom 0.18 to 0.34, and more usually ranging from 0.19 to 0.33, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.33, usually greater than 0.34, and more usually ofgreater than 0.35.

According to a preferred embodiment of this invention any of the probesdesignated with SEQ ID NO 5, 28 and 6 may be used to identify the type1; any of the probes with SEQ ID NO 8 to 12 or 22 to 26 and 32 to 34 maybe used to identify type 2; and any of the probes with SEQ ID NO 13, 14,36, 21, or 54 to identify type 3; and any of the probes with SEQ ID NO17, 18 or 19 and 37 to 43 and probes of SEQ ID NO 49, 50 and 53 toidentify type 4.

Probes 44 to 47 may be used to identify type 5, probe 48 may be used toidentify type 6.

The following regions might also be used for discrimination of certaintypes: the region between positions −238 to −223 for type 2, the regionbetween positions −244 to −229 for type 4, the regions between positions−253 to −238, or between positions −275 to −260, or between positions−293 to −278 for type 3.

The nucleotide at position −2 can also be employed to furtherdiscriminate between certain types or subtypes.

The process of the invention also comprises the discrimination andclassification of subtypes of HCV, wherein besides the above-mentionedprobes also probes hybridizing to the following target regions are used,or said regions wherein T is replaced by U or said regions which arecomplementary to the above-defined regions,

for HCV type 1, CCC CGC AAG ACT GCT A (No. 31) subtype 1a: for HCV type1, CCG CGA GAC TGC TAG C (No. 7) subtype 1b: CCG CGA GAC YGC TAG C (No.30)

wherein Y represents C or T,

for HCV type 2, TTR CCG GTA AGA CTG G (No. 9) subtype 2a: TGR CCG GGCATA GAG T (No. 10) CCG GGA AGA CTG GGT C (No. 22) ACC CAC TCT ATG CCC G(No. 24)

wherein R represents A or G,

for HCV type 2, TTA CCG GGA AGA CTG G (No. 11) subtype 2b: TGA CCG GACATA GAG T (No. 12) CCG GAA AGA CTG GGT C (No. 23) ACC CAC TCT ATG TCC G(No. 25) for HCV type 2, GGA CCC AGT CTT CCT G (No. 33) subtype 2c: TGCCTG GTC ATT TGG G (No. 34) for HCV type 3, AAT CGC TGG GGT GAC C (No.13) subtype 3a: TTT CTG GGT ATT GAG C (No. 14) TKT CTG GGT ATT GAG C(No. 35)

wherein K represents G or T,

for HCV type 3, TTT CCG GGC ATT GAG C (No. 19) subtype 3b: AAT CGC CGGGAT GAC C (No. 38) CCG CGA GAT CAC TAG C (No. 21) for HCV type 3, GAGTGT CGT ACA GCC T (No. 51) subtype 3c: GAA TCG CCG GGT TGA C (No. 54)TTT CCG GGC ATT GAG C (No. 19) CCG CGA GAC TGC TAG C (No. 7) for HCVtype 4, AAT CGC CGG GAT GAC C (No. 38) subtype 4a or 4d: TTT CCG GGC ATTGAG C (No. 19) for type 4, AAT CGC CGG GAT GAC C (No. 38) subtype 4b:AAT GCC CGG CAA TTT G (No. 41) AAT CGC CGG GAC GAC C (No. 40) for type4, AAT CGC CGA GAT GAC C (No. 42) subtype 4c: AAT GCT CGG AAA TTT G (No.43) TGC CTG GAA ATT TGG G (No. 50) GGA ATC GCC AGG ACG A (No. 53) CCGCGA GAC TGC TAG C (No. 7) for type 4, AAT CGC CGG GAC GAC C (No. 40)subtype 4e: GAG TGT TGT GCA GCC T (No. 39) AAT GCC CGG CAA TTT G (No.41) for type 4, subtype TTT CCG GGC ATT GAG C (No. 19) 4f: AAT CGC CGGGAT GAC C (No. 38) GAG TGT CGT ACA GCC T (No. 51) CCG CGA GAC TGC TAG C(No. 7) for type 4, subtype TGC CTG GAA ATT TGG G (No. 50) 4g(provisional): GGA ATC GCC AGG ACG A (No. 53) for type 4, subtype AATCGC CAG GAT GAC C (No. 49) 4h (provisional): TGC CTG GAA ATT TGG G (No.50)

or the probes used are a set of two probes chosen from among the definedprobes.

The invention also relates to the use of the above-defined method fordetermining the HCV subtype(s) present in a biological sample to beanalyzed.

The term “subtype” corresponds to a group of HCV isolates of which thecomplete genome or complete polyprotein shows a homology of more than90% both at the nucleic acid and amino acid levels, or of which theregion in NS5 between nucleotide positions 7935 and 8274 shows ahomology of more than 88% at the nucleic acid level to the correspondingparts of the genomes of the other isolates of the same group, with saidnumbering beginning with the adenine residue of the initiation coding ofthe long ORF. Isolates belonging to different subtypes of HCV andbelonging to the same type of HCV show homologies of more than 74% atthe nucleic acid level and of more than 78% at the amino acid level.

More preferably the above mentioned process relates to classification ofHCV isolates into type and subtypes should be performed according to thefact that,

(1) based on phylogenetic analysis of nucleic acid sequences in the NS5bregion between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and3600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al, 1991),isolates belonging to the same HCV type show nucleotide distances ofless than 0.34, usually less than 0.33, and more usually of less than0.32, and isolates belonging to the same subtype show nucleotidedistances of less than 0.135, usually of less than 0.13, and moreusually of less than 0.125, and consequently isolates belonging to thesame type but different subtypes show nucleotide distances ranging from0.135 to 0.34, usually ranging from 0.14 to 0.33, and more usuallyranging from 0.15 to 0.32, and isolates belonging to different HCV typesshow nucleotide distances greater than 0.34, usually greater than 0.35,and more usually of greater than 0.36,

(2) based on phylogenetic analysis of nucleic acid sequences in thecore/E1 region between nucleotides 378 and 957, isolates belonging tothe same HCV type show nucleotide distances of less than 0.38, usuallyof less than 0.37, and more usually of less than 0.36, and isolatesbelonging to the same subtype show nucleotide distances of less than0.17, usually of less than 0.16, and more usually of less than 0.15, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.15 to 0.38, usually rangingfrom 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.36, usually more than 0.365, and more usually of greaterthan 0.37,

(3) based on phylogenetic analysis of nucleic acid sequences in theNS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) orbetween nucleotides 4993 and 5621 (Kato et al., 1990) or betweennucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging tothe same HCV type show nucleotide distances of less than 0.35, usuallyof less than 0.34, and more usually of less than 0.33, and isolatesbelonging to the same subtype show nucleotide distances of less than0.19, usually of less than 0.18, and more usually of less than 0.17, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.17 to 0.35, usually rangingfrom 0.18 to 0.34, and more usually ranging from 0.19 to 0.33, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.33, usually greater than 0.34, and more usually ofgreater than 0.35.

Using these criteria, HCV isolates can be classified into at least 6types.

Several subtypes can clearly be distinguished in types 1, 2, 3 and 4(1a, 1b, 2a, 2b, 2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e and 40 based onhomologies of the 5′ UR and coding regions including the part of NS5between positions 7935 and 8274 and the C/E1 region between nucleotides317 and 957, and based on comparisons with isolates Z1 and DK13 asdescribed in Bukh et al. (1993).

Further subdivision of type 4 into subtypes 4g and 4h is tentative andonly based on differences in the 5′ UR. An overview of most of thereported isolates and their proposed classification according to thetyping system of the present invention is given in Table 1.

According to a preferred embodiment of the present invention, the probewith SEQ ID NO 31 may be used to identify subtype 1a; the probes withSEQ ID NO 7 and 30 may be used to identify subtype 1b; any of the probeswith SEQ ID NO 9, 10, 22, or 24 may be used to identify subtype 2a; anyof the probes with SEQ ID NO 11, 12, 23, or 25 may be used to identifysubtype 2b; any of the probes with SEQ ID NO 33 or 34 may be used toidentify subtype 2c; any of the probes with SEQ ID NO 13, 14, or 35 maybe used to identify subtype 3a; any of the probes with SEQ ID NO 38, 19and 21 may be used to identify subtype 3b, 4a or 4d; any of the probeswith SEQ ID NO 38 or 41 may be used to identify subtype 4b; any of theprobes with SEQ ID NO 42 or 43 may be used to identify subtype 4c; anyof the probes in SEQ ID NO 39, 40, or 41 may be used to identify: 4e,51, 38, 19, or 7; 4f; any of the probes with SEQ ID NO 49 or 50 may beused to identify the putative subtype 4h; any of the probes with SEQ IDNO 50 or 53 may be used to identify the putative subtype 4g.

According to a preferred embodiment of the process of the invention, theHCV types or subtypes to be discriminated are also identified by meansof universal probes for HCV, such as the ones targeting one of thefollowing regions:

TTG GGC GYG CCC CCG C (No. 20) TCT GCG GAA CCG GTG A (No. 27)

According to another advantageous embodiment of the process of theinvention, the hybridization step is preceeded by an amplification stepof the deoxyribonucleotide or ribonucleotide containing the region totarget, advantageously comprising the following steps:

-   -   contacting the biological sample liable to contain the isolate        to be typed or subtyped with a set of primers, flanking the        region to target, with said primers being complementary to        conserved regions of the HCV genome, and preferably primers        being complementary to the 5′ untranslated conserved regions of        the HCV genome, with said primers preferably having at least 8        contiguous nucleotides more preferably about 15, and even more        preferably more than 15 contiguous nucleotides, with said        contiguous nucleotides being respectively complementary to        sequences chosen from the region extending from nucleotide −341        to nucleotide −171 and from the region extending from nucleotide        −67 to nucleotide −1, of FIGS. 2 and 4.

Alternatively, the antisense primers could also extend into the coreregion or the set of primers may or/be aimed at amplifying both the 5′URand the core region, either in 1 PCR fragment or with a set of primersfor each of the two regions. Consequently, probes from the core region,able to hybrize to (sub)type specific regions in core PCR products, maybe included in the line probe assay to further discriminate betweentypes and/or subtypes,

-   -   amplifying the target region, for instance via a polymerase        chain reaction by means of the above-mentioned set of primers        and possibly incorporating a label such as digoxigenin or biotin        into the amplified target sequence, with said amplifying being        repeated between 20 and 80 times, advantageously between 30 and        50 times.

According to a preferred embodiment of the invention, the analyte strandmay be enzymatically or chemically modified either in vivo or in vitroprior to hybridization. Many systems for coupling reporter groups tonucleic acid compounds have been described, based on the use of suchlabels as biotin or digoxigenin. In still another embodiment of theinvention sandwich hybridization may be used. In a preferred embodiment,the target sequence present in the analyte strand is converted intocDNA, with said cDNA being amplified by any technique known in the artsuch as by the polymerase chain reaction (PCR; Saiki et al., 1988),ligase chain reaction (LCR; Landegren et al., 1988; Wu & Wallace, 1989;Barany, 1991), nucleic acid sequence-based amplification (NASBA;Guatelli et al., 1990; Compton, 1991), transcription-based amplificationsystem (TAS; Kwoh et al., 1989), strand displacement amplification (SDA;Duck, 1990; Walker et al., 1992) or amplification by means of Qbreplicase (Lizardi et al., 1988; Lomeli et al., 1989).

The cDNA amplification step is preferably achieved by means of PCRtechnology and may consist of steps:

(a) providing a set of primers for a polymerase chain reaction methodwhich flank the target sequence to be detected;

(b) amplifying the target region via a polymerase chain reaction methodby means of the primers of (a); and in the same step an appropriatelabel molecule can be incorporated into the amplified target said labelmolecule being preferably digoxigenin or biotin.

The term “primers” corresponds to oligonucleotide sequences beingcomplementary to conserved regions of sense or antisense strands of cDNAor RNA derived from the HCV genome; preferably of the 5′ untranslatedconserved regions of the HCV genome and more preferably selected fromconserved regions of the 5′ untranslated region of the HCV genomecomprising positions −341 to −171 and −67 to −1, or the core region.

In an advantageous embodiment of the invention, the process is such thatamplification consists of a double PCR step, each step involving aspecific set of primers, with the said first step involving outerprimers selected from the region extending from nucleotide −341 tonucleotide −186 and from the region extending from nucleotide 0.52 tonucleotide −1, and more particularly the following set:

CCC TGT GAG GAA CTW CTG TCT TCA CGC (No. 1) GGT GCA CGG TCT ACG AGA CCT(No. 2)

or their complements,

wherein W represents A or T, and with the second step involving nestedprimers selected from the region extending from nucleotide −326 tonucleotide −171 and from the region extending from nucleotide −68 tonucleotide −1 and, more particularly the following set:

TCT AGC CAT GGC GTT AGT RYG AGT GT (No. 3) CAC TCG CAA GCA CCC TAT CAGGCA GT (No. 4)wherein R represents A or G and Y represents T or C

or their complements.

According to this embodiment of the invention, a double PCR is performedwith outer primers in the first round including sequences as shown inSEQ ID NO 1 and 2, or their complementary sequences and with nestedprimers for the second round including sequences as shown in SEQ ID NO 3and 4, or their complementary sequences.

The term “appropriate label molecule” may include the use of labelednucleotides incorporated during the polymerase step of the amplificationsuch as illustrated in Saiki et al. (1988) and Bej et al. (1990) and orany other method known to the person skilled in the art.

The assays as described in this invention may be improved in severalways obvious for the person skilled in the art. For example the cPCRreactions can be preceded by an RNA-capture step.

According to yet another embodiment, the present invention relates to acomposition comprising at least one oligonucleotide primer, with saidprimers preferably having at least 15 contiguous nucleotides, with saidcontiguous nucleotides being respectively complementary to sequenceschosen from the region extending from nucleotide −341 to nucleotide −171and from the region extending from nucleotide −67 to nucleotide −1 (, ofFIG. 2), or their complement.

According to yet another embodiment, the present invention relates to acomposition comprising at least one oligonucleotide primer preferablyhaving at least 15 contiguous nucleotides, with said contiguousnucleotides being chosen from any of the following sequences:

CCC TGT GAG GAA CTW CTG TCT TCA CGC (No. 1) GGT GCA CGG TCT ACG AGA CCT(No. 2) TCT AGC CAT GGC GTT AGT RYG AGT GT (No. 3) CAC TCG CAA GCA CCCTAT CAG GCA GT (No. 4)wherein W represents A or T, R represents A or G, and Y represents T orC,

or their complements.

According to an advantageous embodiment, the process of the inventionfor the simultaneous typing of all HCV isolates contained in abiological sample comprises the step of contacting one of the followingelements:

-   -   either said biological sample in which the genetic material is        made available for hybridization,    -   or the purified genetic material contained in said biological        sample,    -   or single copies derived from the purified genetic material,    -   or amplified copies derived from the purified genetic material,        with a solid support on which probes as defined above, have been        previously immobilized.

According to this preferred embodiment of the invention, the probes asdefined above are immobilized to a solid substrate.

The term “solid substrate” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate or a membrane (e.g. nylon or nitrocellulose).

Prior to application to the membrane or fixation it may be convenient tomodify the nucleic acid probe in order to facilitate fixation or improvethe hybridization efficiency. Such modifications may encompasshomopolymer tailing, coupling with different reactive groups such asaliphatic groups, NH₂ groups, SH groups, carboxylic groups, or couplingwith biotin or haptens.

According to an advantageous embodiment of the invention, the processcomprises the step of contacting anyone of the probes as defined above,with one of the following elements:

-   -   either a biological sample in which the genetic material is made        available for hybridization,    -   or the purified genetic material contained in said biological        sample,    -   or a single copy derived from the purified genetic material,    -   or an amplified copy derived from the purified genetic material,        with said elements being previously immobilized on a support.

The invention also relates to the typing of new isolates.

More particularly the invention relates to a process for the detectionand identification of novel HCV types or subtypes different from theknown types or subtypes and comprising the steps of:

-   -   determining to which known types or subtypes the HCV isolate        present in the biological sample belongs to, according to the        process as defined above, possibly with said biological sample        being previously determined as containing HCV, possibly by means        of HCV antigen or antibody assays or with a universal probe for        HCV, such as those defined above,    -   in the case of observing a sample which does not hybridize        positively with at least one of the probes able to target the        regions chosen from any of the two domains as defined above,        sequencing the complete genome of the HCV type present in the        sample, or alternatively sequencing that (the) portion(s) of the        5′ untranslated region of the sample corresponding to a new type        and/or subtype to be determined.

Advantageously the process for the detection and identification of novelHCV types and/or subtypes, present in a biological sample, which aredifferent from type 1, type 2, type 3, type 4, type 5, type 6, in thecase of identifying a novel type; and which are different from subtypes1a and 1b for a type 1 HCV isolate, from subtypes 2a, 2b, and 2c for atype 2 isolate, from subtypes 3a, 3b and 3c for a type 3 isolate, fromsubtypes 4a, 4b, 4c, 4d, 4e, 4f, 4g and 4h for a type 4 isolate; fromsubtype 5a for a type 5 isolate; from subtype 6a for a type 6 isolate,in the case of identifying a novel subtype, and comprising the steps of:

-   -   determining to which known type(s) or subtype(s) the HCV        isolate(s) present in the biological sample to be analyzed        belongs, according to the process of the invention, possibly        with said biological sample being previously determined as        containing HCV, possibly by means of HCV antigen or antibody        assays or with a universal probe for HCV such as the one defined        above,    -   in the case of observing a sample which does not hybridize to at        least one of the probes able to target the regions chosen from        any of the type specific or subtype specific domains as defined        above, more particularly not hybridizing with SEQ ID NO 5, 28        and 6 for type 1, with SEQ ID NO 8 to 12 or 22 to 26 and 32 to        34 for type 2, with SEQ ID NO 13, 14, 36, 21 or 54 for type 3,        and with SEQ ID NO 17, 18, 19, 37 to 43, 49, 50 and 53 for type        4; and with SEQ ID NO 7 and 30 for subtype 1b, with SEQ ID NO 31        for subtype 1a, with SEQ ID NO 9, 10, 22 or 24 for subtype 2a,        with SEQ ID NO 11, 12, 23 or 25 for subtype 2b, with SEQ ID NO        33 or 34 for subtype 2c, with SEQ ID NO 13, 14 or 35 for subtype        3a, with SEQ ID NO 38, 21 and 19 for subtype 3b, 4a or 4d, with        SEQ ID NO 38 or 41 for subtype 4b; with SEQ ID NO 42 or 43 for        subtype 4c; with SEQ ID NO 39, 40, or 41 for subtype 4e, with        SEQ ID NO 51, 38, 19 or 7; for subtype 4f; with SEQ ID NO 49 or        50 for the putative subtype 4h; with SEQ ID NO 50 or 53 for the        putative subtype 4g, sequencing the complete genome of the HCV        type present in the sample, or, alternatively sequencing that        (the) portion(s) of the 5′ untranslated region of the sample        corresponding to a new type and/or subtype to be determined.

The term “new isolates” corresponds to isolates which are not able tohybridize to any of the 9 above-mentioned regions or show reactivitieswhich cannot be correctly interpreted as matching one of the currentlyknown HCV types or subtypes. This special embodiment of the inventionmay also be performed by the steps of:

(a) screening HCV antibody-positive sera, or clinical NANB hepatitissamples, or a population of random samples, by cPCR (cDNA PCR),

(b) performing a HCV LiPA with those samples from which a cPCR producthas been obtained, and

(c) cloning and sequencing these PCR fragments showing aberrantreactivities.

The invention also relates to a method for determining the type(s) aswell as the subtypes(s) of HCV, and/or HIV, and/or HBV and/or HTLVpresent in a biological sample, which comprises the steps of:

-   -   providing:        -   at least one of the probes as defined above, preferably the            probes as defined above, enabling the genotyping (typing            and/or subtyping) of HCV, and at least one of the following            probes:        -   probes capable of detecting oligonucleotides of HIV types 1            and/or 2 which can be present in said biological sample,            and/or        -   probes capable of detecting oligonucleotides of HBV            subtypes, and/or sAg mutants, and/or cAg mutants which can            be present in said biological sample, and/or        -   probes capable of detecting oligonucleotides of HTLV-I            and/or HTLV-II suspected to be in the biological sample,    -   possibly providing a set of primers as defined above, as well at        least one of the following primers: sets of primers to        respectively amplify human immunodeficiency virus (HIV), and/or        HBV and/or human T-cell lymphotropic virus (HTLV)        oligonucleotides, by means of PCR reaction and amplifying the        oligonucleotides of HCV, and either HBV and/or HIV and/or HTLV        possibly present in the biological sample,    -   contacting        -   the biological sample in which the genetic material is made            available for hybridization,        -   or the purified genetic material contained in said            biological sample,        -   or single copies derived from the purified genetic material,        -   or amplified copies derived from the purified genetic            material,

with the above-mentioned probes defined above under conditions whichallow hybridization between the probes and the target sequences ofisolates of HCV and at least one of the following viruses: HBV, and/orHIV, and/or HTLV,

-   -   detecting the complexes possibly formed between the probes used        and the target regions possibly present in the biological        sample.

According to this embodiment, in addition to the type or subtype of HCVpresent in a biological sample, the invention also relates to a methodfor determining the type or subtype of any other parenterallytransmitted viral isolate such as HTLV, HIV, HBV characterized byincorporating on one and the same strip, probes hybridizing specificallyto:

-   -   the different types and/or subtypes of HCV as defined above,    -   human immunodeficiency viruses HIV-1 and HIV-2,    -   human T-cell lymphotrophic viruses HTLV-1 and HTLV-2,    -   the different HB surface antigen (HBsAg) mutants or HB core        antigen (HBcAg) or HB precore Ag mutants.

In some test samples, different target sequences of which the specificdetection is of clinical relevance are present simultaneously. For eachof these target sequences a separate hybridization test with thecorresponding probe should be performed. The combination of differenttype/subtype specific probes comprised in the art, in combination withthe new and inventive HCV type/subtype-specific probes as explained inthe present invention on one membrane strip could provide an easy andreliable general typing system for parenterally transmitted human viraldiseases. If analyte strand amplification is necessary, a set of primerscan be provided per viral organism to be differentiated and classified.

The invention also relates to a solid support, particularly a membranestrip containing, on known locations of its surface, a selection of thefollowing probes, or their complements, or the above-mentioned probeswherein T has been replaced by U:

-   -   NO 5, NO 6, NO 7, NO 8, NO 9, NO 10, NO 11, NO 12, NO 13, NO 14,        NO 15, NO 16, NO 17, NO 18, NO 19, NO 20, NO 21, NO 22, NO 23,        NO 24, NO 25, NO 26, NO 27, NO 28 to 54 and NO 93 to 96, as        defined above, as well as a control to determine if there is        hybridization between these probes and the ribo or        deoxyribonucleotide strands of HCV, liable to be contained in a        biological sample in which HCV isolates are to be        differentiated.

According to a specially preferred embodiment of the invention, theprobes are immobilized in a line-wise fashion to a membrane strip.

In this preferred embodiment of the invention, a set of probes eachapplied to a known location onto the membrane strip include probesselected from the sequences with SEQ ID NO 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, SEQ ID NO 28to 54, and NO 93 to 96 as well as a control line for conjugate binding.

The method of this preferred embodiment of the invention makes itpossible to quickly determine the type of HCV infection. This assayprovides the ability to discriminate between at least 6 different HCVtypes and might discriminate between at least 18 subtypes, and is a goodinstrument for searching for new types or (sub)types of HCV. Forexample, new subtypes 1c, Id and type 7, and other new (sub)types maycontain specific mutations in the regions mentioned above, which can beemployed for specific detection by means of type-specific probes derivedfrom such new sequences.

The invention also relates to a kit for the in vitro discrimination ofat least one HCV isolate from a biological sample liable to contain it,and for its classification it according to the HCV type and subtype,with said kit containing

-   -   a least one probe selected among any of those defined above;    -   a buffer or components necessary for producing the buffer        enabling hybridization reaction between these probes and the        cDNAs and/or RNAs of HCV isolates to be carried out;    -   when appropriate, means for detecting the hybrids resulting from        the preceding hybridization.

The invention also relates to a kit for typing at least one HCV isolatefrom a biological sample liable to contain it and for classifying itaccording to the HCV type and subtype, with said kit containing

-   -   possibly one universal probe as defined above,    -   at least one probe selected among any of those of the invention,    -   a buffer or components necessary for producing the buffer        enabling hybridization reaction between these probes and the        DNAs and/or RNAs of HCV isolates to be carried out;    -   when appropriate, means for detecting the hybrids resulting from        the preceding hybridization.

According to this embodiment, the invention also relates to a kit forgenotyping (typing and/or subtyping) of HCV isolates comprising:

-   -   a set of probes as defined above, preferentially immobilized on        a solid substrate, and more preferentially on one and the same        membrane, and    -   possibly a set of primers as defined above,    -   a set of buffers necessary to carry out the hybridization as        well as the detection of the hybrids formed.

The invention also relates to a kit for typing HCV isolates belonging toat least one of the following HCV types: HCV type 1, HCV type 2, HCVtype 3, HCV type 4, HCV type 5, HCV type 6 with said kit containing atleast one of the probes as above defined,

-   -   the buffer or components necessary for producing the buffer        enabling hybridization reaction between these probes and the        cDNAs and/or RNAs of the above-mentioned HCV isolates to be        carried out;    -   when appropriate, means for detecting the hybrids resulting from        the preceding hybridization.

The invention advantageously relates to a kit for the discrimination andclassification of HCV types and subtypes, with said kit containing:

-   -   at least one of the probes as defined above,    -   the buffer or components necessary for producing the buffer        enabling hybridization reaction between these probes and the        DNAs and/or RNAs of the above-mentioned HCV isolates to be        carried out;    -   when appropriate, means for detecting the hybrids resulting from        the preceding hybridization.

It is to be mentioned that all the probes from SEQ ID NO 1 to SEQ ID NO54 and SEQ ID NO 93 to 96 are new.

Furthermore, probes of SEQ ID NO 18, 29, 33, 34, 35, 40, 42, 43, 47, 49and 54 are derived from new sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Ethidium bromide-stained agarose gel showing the length of the nestedPCR fragments. Lane A of each pair shows the PCR fragment withincorporation of Bio-11-dUTP. Lane B is the PCR fragment withoutBio-11-dUTP. 1: Serum BR28, 2: Serum BR24, 3: Serum BR29, 4: Serum BR33,5: Serum BR36, 6 and 7: negative control sera, 8: Serum JP62, 9: SerumBR23, 10: cPCR control without template, M: molecular weight markers.

FIG. 2

Alignment of the 5′ UR nucleotide sequences of isolates from fourdifferent types of HCV. Boxed nucleotides indicate the positions ofprobes used for typing of the four different groups. The underlinednucleotides are used for subtyping within each group. The period betweennucleotide −140 and −139 in most sequences corresponds to the insertionin some of the type 4 isolates. The numbering of the probes correspondswith the numbers used in Table 4.

FIG. 3

HCV LiPA Typing results of some representative sera. The strip contains19 parallel probe lines:

A: Probe 5 (SEQ ID NO 5); B: Probe 6 (SEQ ID NO 6); C: Probe 7 (SEQ IDNO 7); D: Probe 8 (SEQ ID NO 8); E: Probe 26 (SEQ ID NO 26); F: Probe 22(SEQ ID NO 22) and Probe 24 (SEQ ID NO 24); G: Probe 10 (SEQ ID NO 10);H: Probe 13 (SEQ ID NO 13); I: Probe 14 (SEQ ID NO 14); J: Probe 21 (SEQID NO 21); K: Probe 15 (SEQ ID NO 15); L: Probe 16 (SEQ ID NO 16); M:Probe 17 (SEQ ID NO 17); N: Probe 19 (SEQ ID NO 19); 0: Probe 18 (SEQ IDNO 18); P: Probe 155 (antisense probe: 5′-GGGGGCCTGGAGGCTG-3′) (SEQ IDNO 97); Q: Probe 27 (SEQ ID NO 27); R: Probe 20 (SEQ ID NO 20); S:control line for conjugate binding.

The strips were hybridized with cPCR products of the following sera:Strip 1: serum BR5, Strip 2: serum BR12, Strip 3: serum BR18, Strip 4:serum BR22, Strip 5: serum BR19, Strip 6: serum BE95, Strip 7: serumBU79, Strip 8: serum BR23, Strip 9: serum JP63.

FIG. 4

Nucleotide sequence alignment of the HCV 5′ untranslated regions of newisolates BE90, BE91, BE92, BE93, BE94, BE95, BE96, BE97, BE98, BE99,GB48, GB116, GB358, GB569, GB549, GB809, CAM600, CAM736, GB478, GB724,and GB438, with sequences of HCV type 1a (HCV-1), 1b (HCV-J), 2a(HC-J6), 2b (HC-J8), 3a (BR56), 3b (HCV-TR), 5 (SA1), 6 (HK1). Thesequences used to construct this alignment are taken from the EMBLdatabase and have the following accession number: ¹ M62321, ² D10749, ³D00944, ⁴ D01221, ⁵ D13448, ⁶ D11443, ⁷ M84838, and 8 L08156. Thesequences between nucleotides −220 and −180 are not shown, they areidentical to HCV-I in all isolates. ‘-’, nucleotide is identical to thecorresponding nucleotide in HCV-1; ‘..’, gap created between −145 and−144 to allow alignment with type 6 sequences which have a CA insertion;‘.’, gap created between −138 and −137 in most of the sequences topreserve alignment with sequences which have an extra nucleotide at thatposition. * refers to the conserved HCV sequence between residues −220and −180 as shown in FIG. 2.

FIG. 5

Amino acid sequence alignment of the NS5 sequences of isolates BE90,BE91, BE92, BE93, BE95, GB358, GB549, and GB809 with known sequences asdescribed in Table 6.

FIG. 6

Line probe assays including probe with SEQ ID NO 32, tested with type 1and 2 sera. 1, type 1b serum BE82, 2, type 2a serum JP62, 3, type 2bserum BE91, A, conjugate control, B, probes 20 and 27, C, probe 8, D,probe 26, E, probe 32 (SEQ ID NO 32).

FIG. 7

Line probe assays including probes with SEQ ID NO 33 and 34, tested withtype 2a, 2b, and 2c sera. 1, type 2a serum JP62, 2, type 2b serum BE91,3, type 2c serum BE92, A, conjugate control, B, probes 20 and 27, C,probe 8, D, probe 26, E, probe 32, F, probe 22, G, probe 24, H, probe23, I, probe 25, J, probe 33 (SEQ ID NO 33), K, probe 34 (SEQ ID NO 34).

FIG. 8

Line probe assays including probes with SEQ ID NO 31, 37 and 38, testedwith type 4 sera. 1, type 4a serum GB116, 2, serum GB113, 3, type 4fserum GB438, A, conjugate control, B, probes 20 and 27, C, probe 37 (SEQID NO 37), D, probe 38 (SEQ ID NO 38), E, probe 19, F, probe 31 (SEQ IDNO 31), G, probe 7.

FIG. 9

Line probe assays including probes with SEQ ID NO 44, 45 and 46, testedwith type 4a and 5a sera. 1, type 5a serum 3E95, 2, type 4a serum GB116,A, conjugate control, B, probes 20 and 27, C, probe 44 (SEQ ID NO 44),D, probe 45 (SEQ ID NO 45), E, probe 46 (SEQ ID NO 46), F, probe 31 (SEQID NO 31), G, probe 7.

FIG. 10

Line probe assays including probes with SEQ ID NO 93, 94, 95, and 96,tested with type 4a and 5a sera. 1, type 4a serum GB116, 2, type 5aserum BE95, A, conjugate control, B, probes 20 and 27, C, probe 93 (SEQID NO 93) applied at a concentration of 0.4 pmol/μl, D, probe 94 (SEQ IDNO 94) applied at a concentration of 2.5 pmol/μl, E, probe 94 (SEQ ID NO94) applied at a concentration of 1.0 pmol/μl, F, probe 94 (SEQ ID NO94) applied at a concentration of 0.4 pmol/μl, G, probe 95 (SEQ ID NO95) applied at a concentration of 2.5 pmol/μl, H, probe 95 (SEQ ID NO95) applied at a concentration of 1.0 pmol/μl, I, probe 95 (SEQ ID NO95) applied at a concentration of 0.4 pmol/μl, J, probe 96 (SEQ ID NO96) applied at a concentration of 0.4 pmol/μl.

TABLE 1

Overview of the different classification systems.

Table 2

Interpretation of the results shown in FIG. 3.

Table 3

Final results of HCV LiPA typing and HCV antibody assays.

A summary of the typing in relation to the serology is presented. TheINNO-LIA HCV Ab assay contains one line with NS4 epitopes, one line withNS5 epitopes, and 4 lines with core epitopes. Only the highest score forthe core lines is given. The intensity of the signal is given by anumber: 0=negative; 9=indeterminate; 1 to 3=positive. The finalinterpretation of the antibody test is given in the LIA column:1=positive; 0=negative; 9=indeterminate.

The signal-to-noise ratio of the sera tested in the Innotest HCV Ab isalso given for some of the sera.

Table 4

Nucleotide sequence, position, and orientation of the primers andprobes.

Table 5

Overview of new probes designed from new types and subtypes of HCV. Thetype or subtype for which classification is possible or improved, thesequence, and the SEQ ID NO. are shown.

* represents a probe which does not type or subtype all isolates foundrepresenting said type or subtype. The underlined letters indicateprovisional divisions into subtypes.

Table 6

Sequence homology between BE90, BE91, BE92, BE93, BE95, GB358, GB549,and GB809 and published sequences in the HCV NS5 region from nucleotide7935 to 8274, according to the numbering used in the present invention.Homology scores within the same subtype are in bold. Published sequencesused to perform homology calculations were taken from the EMBL databaseand have the following accession numbers: M62321, ² D10749, ³ M67463, ⁴D90208, ⁵×61596, ⁶ L02836, ⁷ M84754, ⁸ D10750, ⁹ D11168; D01171, ¹⁰S38204, ¹¹ M58335, ¹² D10078, ¹³ D10079, ¹⁴ D10080, ¹⁵ D10081, ¹⁶D00944, and ¹⁷ D01221. All of them represent complete genomes, except¹², ¹³, ¹⁴, ¹⁵ and ¹⁸ for which NS5 sequences were published. ¹⁸ waspublished in the Chiron patent WO 92/19743, SEQ ID NO 18.

EXAMPLES

In order to study the natural variation of HCV isolates obtained fromdifferent geographical areas throughout the world, a rapid means fortyping and subtyping of HCV isolates in the form of a Line Probe Assay(LiPA) was developed.

Essentially, a cPCR fragment containing incorporated biotinylated dUTPis hybridized to oligonucleotides which are immobilized on anitrocellulose membrane. The stable hybridization duplex is thenrevealed by streptavidin-labelled alkaline phosphatase, and subsequentcolor development with NBT (nitro 42 blue tetrazolium) and BCIP(bromochloro-indolyl phosphate). The cPCR fragment is synthesized fromthe 5′ UR of any HCV RNA using highly conserved sets of primers. Theoligonucleotides used for typing are directed against the internaltype-specific variable parts of the cPCR fragment. In fact, the 2variable regions between positions −170 and −155, and between −132 and−117 in the linear sequence may be part of a stem in the folded viralRNA, and mutations in the first region may be complemented by anothermutation in the second region to allow or disallow RNA duplex formation.Variation and conservation is expected to occur at the same positions inother new types of HCV as well and, therefore, this variable regionmight remain instrumental for the discrimination between all current andyet-to-be discovered types of HCV. Moreover, since higher variabilitiescompared to the 5′ UR are observed in the core, NS3, and NS5 regions,typing in these regions employing universal sets of primers might nolonger be tenable.

The proposed nomenclature of this invention is provisional and couldstill be subject to amendments according to new guidelines that may beset forward by international committees. For example, subtype 4a mightbe changed into another type 4 subtype, like 4c or 4e, and type 4 mightbe changed into type 5 or 6, in which case type 4a might become 6c, forexample. However, new classification systems will not hamperclassification of a certain group of isolates classified into a type orsubtype by means of the proposed probes of the invention.

1. Serum Samples Used for Typing and Subtyping

Sixty-one Brazilian samples (BR1 to BR61) were tested in the HCVAntibody ELISA assay (Innotest HCV Ab, Innogenetics) as well as in theInno-LIA HCV Ab test (Innogenetics). The first 23 serum samples (BR1 toBR23, Table 3) were taken from hemodialysis patients with either highALT levels or positive Inno-LIA results, or from blood donors from whichthe recipient developed NANB hepatitis liver disease. Fourteen (BR24 toBR37) of the other serum samples were randomly chosen; the 24 remainingsera (BR38 to BR61) were selected on the basis of their LIA pattern.Most of the latter showed weakly positive, indeterminate, or negativereactivity with the NS4 and NS5 synthetic peptides on the LIA. Thefollowing sera were also included in this typing effort: two pools ofJapanese sera (JP62 and J63), six Belgian sera (BE64 to BE69), four serafrom the Netherlands (NE70 to NE73), six sera from Burundi (BU74 toBU79) and two sera from Gabon (GB80 and GB81). They were all tested withthe Inno-LIA HCV Ab assay system. The sera BU74 to BU78 were onlypositive for anti-core antibodies, while the serum BU79 reacted onlywith the NS5 line. Both Gabonese sera were LIA HCV negative (Inno-LIAHCV), HIV negative (Innotest HIV), but HTLV positive (Innotest HTLV).One serum from Belgium (BE69) and one from the Netherlands (NE73) werecompletely negative. Three of the NF-sera (NE71 to NE73) were selectedbecause they were negative in the second generation RIBA test (OrthoDiagnostics Inc.).

2. cPCR, Analysis of the PCR Product, and Cloning

The primers used for the PCR reactions were complementary to theconserved areas of the 5′ UR of the different HCV types. Degenerationwas included to allow annealing to type 1 and type 2 sequences (Kato etal, 1990; Nakao et al, 1991; Okamoto et al., 1991) and to the sequenceof our type 3 clone (BR56; accession number D13448, DDJJB/EMBL/GenBankDNA data base deposited on 21/10/1992). The sequences of the outer PCRprimers (HcPr98, SEQ ID NO 1 and HcPr29, SEQ ID NO 2) and of the nestedPCR primers (HcPr95, SEQ ID NO 3 and HcPr96, SEQ ID NO 4) are listed inTable 4. The probes used for the detection of the different serum typesare also listed in Table 4. All oligonucleotides were synthesized on a392 DNA/RNA Synthesizer (Applied Biosystems).

Viral RNA was extracted from serum essentially as described byChomczynski and Sacchi (1987) with minor modifications. The RNA wascoprecipitated with 20 μg Dextran T500 (Pharmacia). The RNA pellet wasbriefly dried and resuspended in 10 μl DEPC-treated H₂O. After adding 2μl 150 ng/μl random primers (Pharmacia) and denaturatine for 10 minutesat 65° C., the first strand cDNA synthesis was carried out in 20 μl at42° C. in the presence of 25 U HPRI (Amersham), 500 μM dATP, dCTP, dTTPand dGTP, 1×AMV buffer (Stratagene) and 2.5 U AMV-RT (Stratagene). Sevenμl of the resulting cDNA was amplified in an outer PCR over 40 cycleseach consisting of 1 min 95° C., 1 min 55° C. and 1 min 72° C. in atotal volume of 50 μl. The solution was adjusted to a finalconcentration of 200 μM of dATP, dCTP, dTTP and dGTP, 1×Taq buffer(Stratagene), 0.2 μM of each primer, and 1 U Taq polymerase(Stratagene). One p. 1 of the first round amplification product wasamplified with the nested primers again for 40 cycles in a buffer withthe same composition. For HCV typing, the nested PCR contained 40 μMBio-11-dUTP (Sigma) and 160 μM of dTTP. Both the outer and the nestedPCR product were then subjected to electrophoresis in a 2% low meltingpoint (NuSieve GTG, FMC)/1% Ultra Pure (Gibco BRL) agarose gel. Afterethidium bromide staining, PCR fragments were cut out from the agarosegel, the DNA was recovered by centrifugation through a 0.45 μm HVmembrane (Millipore), purified by two phenol/chloroform and two etherextractions, precipitated, and subsequently polished with T4 DNApolymerase (Boehringer), kinated with T4 kinase (Boehringer), andfinally ligated in the dephosphorylated Eco RV site of pBluescript KS(−)(Stratagene). Plasmid DNA preparation was as described in the alkalinelysis method (Maniatis et al., 1982). Sequencing reactions were carriedout on double-stranded plasmid DNA with T7 and T3 primers by using theDeaza G/A T7 sequencing mixes (Pharmacia).

The results of these sequencing reactions are shown in FIG. 2. Thefollowing sequences were deposited in DNA databases (BR56:DDBJ/EMBL/Genbank accession number D13448; BU74: DDBJ/EML/GenBank,accession number D13449; BU79: accession number D13450; GB80: accessionnumber D13451; GB81: accession number D13452; GP62: accession numberD13453).

Serum RNA from HCV-infected patients was used as template for cDNAsynthesis, which in turn was a template for nested PCR. Two sets of PCRprimers were designed: HcPr98 (SEQ ID NO 1) and HcPr29 (SEQ ID NO 2) forthe outer reaction, HcPr95 (SEQ ID NO 3) and HcPr96 (SEQ ID NO 4) forthe nested reaction (Table 4). These four primers were chosen to matchthe published sequences (Kato et al., 1990; Nakao et al., 1991; Okamotoet al., 1991) and the sequence of a clone obtained from the untranslatedregion of isolate BR56. (see FIG. 2). The resulting amplificationproduct of the nested PCR is 235 base pairs (bp) long. Due to theincorporation of Bio-11-dUTP, there is a decrease in mobility which isclearly visible after agarose gel electrophoresis (FIG. 1). The size ofthe DNA fragments is the same for all the different HCV types,suggesting that a second experiment, like restriction enzyme digestionor hybridization, is necessary for classification. A membrane stripcontaining immobilized HCV-specific oligonucleotide probes applied asparallel lines was therefore developed. These strips are hybridized withPCR amplified DNA fragments of the 5′ UR into which biotinylatednucleotides were incorporated during synthesis. After hybridization,streptavidin labelled with alkaline phosphatase is added and becomesbound to the biotinylated hybrids formed during the hybridization. Afterincubation with NBT/BCIP, a purple precipitate appears.

3. Preparation of the Line Probe Assay (LiPA) Strips

The 16-mer oligonucleotides, specific for the different types orsubtypes of HCV (Table 4, number 5 to 27), were provided with apoly-(dT) tail at their 3′ end as follows: 20 pmol of primer wasincubated in 25 μl buffer containing 3.2 mM dTTP, 25 mM Tris.HCl (pH7.5), 0.1 M sodium cacodylate, 1 mM CoCl₂, 0.1 mM dithiothreitol, and 60U Terminal deoxynucleotidyl Transferase (Pharmacia) for 1 hour at 37° C.The reaction was stopped by adding 2.5 μl 0.5 M EDTA (pH 8.0) anddiluted with 20×SSC (Maniatis et al., 1982) until a final concentrationof 6×SSC and 2.5 pmol oligonucleotide/μl was reached.

One pmol of this solution was applied over a distance of 4 mm on anitrocellulose membrane. As control for the conjugate, biotinylated DNAwas applied alongside. The oligonucleotides were fixed to the membraneby baking at 80° C. for two hours. The membrane was then sliced in 4-mmstrips.

4. LiPA Test Hybridization and Color Development

Ten μl of the nested PCR amplification product, containing incorporatedBio-11-dUTP, is mixed with 10 μl of 400 mM NaOH/10 mM EDTA and incubatedat room temperature (RT) for 10 minutes. Then, 1 ml prewarmed (37° C.)hybridization buffer containing 3 M tetramethylammonium chloride (TMACl,Merck), 50 mM sodium phosphate (pH 6.8), 1 mM EDTA, 5×Denhardts(Maniatis et al., 1982), 0.6% (w/v) SDS and 100 μg/ml sheared salmonsperm DNA is added and the hybridization is carried out in a shakingwater bath at 42° C. for 2 hours (Jacobs et al., 1988). The strips arewashed 2 times at RT for 5 minutes with 1 ml prewarmed (37° C.) washbuffer (3 M TMACl, 0.2% SDS, 50 mM Tris.HCl, pH 8.0), followed by astringent wash at 51° C. for 30 minutes and two brief washing steps atRT. At this moment, the wash buffer is replaced by Rinse Solution(phosphate buffer containing NaCl, Triton, 0.5% NaN₃; Inno-Lipa,Innogenetics, Antwerp, Belgium) and the strips are rinsed twice with 1ml at RT. Finally, the strips are rinsed with Conjugate Diluent(phosphate buffer containing NaCl, Triton, protein stabilizers, 0.1%NaN₃; Inno-Lia, Innogenetics, Antwerp, Belgium) and incubated withConjugate Diluent containing 4000× diluted streptavidin, labelled withalkaline phosphatase (Gibco BRL) for another 30 minutes at RT. Thestrips are washed again 3 times with Rinse Solution and once withSubstrate Diluent (Tris buffer containing NaCl and MgCl₂; Inno-Lia,Innogenetics, Antwerp, Belgium). Color development is achieved by addingBCIP and NBT to the Substrate diluent and incubation of the strips for30 minutes at RT. The color development is stopped by replacing thebuffer with distilled water.

5. A LiPA for Discrimination Between HCV Types 1, 2 and 3

The sequences for the probes against type 3 were derived from a cPCRclone from serum BR56 (accession number D13448). When comparing thepublished type 1 sequences with BR56, two regions of 16 nucleotidescontaining 4 to 6 mutations could be observed each time. Surprisingly,when type 2 sequences became available, variation was again maintainedin these two regions. Therefore, the position of the typing probes waschosen in those regions with a relatively low degree of homology betweentypes, but good conservation within one type. In a first version of thestrips, a total of eight separately immobilized oligonucleotides wereapplied. Two of them were directed against type 1 (HcPr124, SEQ ID NO 5and HcPr125, SEQ ID NO 6), four against type 2 (HcPr136, SEQ ID NO 9 andHcPr137 (SEQ ID NO 10) for type 2a, HcPr126 (SEQ ID NO 11) and HcPr127(SEQ ID NO 12) for type 2b) and two against type 3 (HcPr128, SEQ ID NO13 and HcPr129, SEQ ID NO 14) HCV (Table 4).

cPCR products were synthesized from 23 Brazilian sera (BR1 to BR23) and,after hybridization, 17 of them recognized the 16-mers of type 1. Fourtype 3 sera were found, as well as one type 2a serum. Serum BR23 wasco-infected with type 1 and type 3. Two pools of Japanese sera weresubsequently tested: JP63 reacted with the type 1 and type 2a probes,and the majority of the JP62 pool contained type 2a sequences. AftercPCR cloning and sequencing the region between the primers HcPr95 (SEQID NO 3) and HcPr96 (SEQ ID NO 4), the sequence of JP62 (FIG. 2) wasconfirmed as type 2a. The type 2b probes HcPr126 (SEQ ID NO 11) andHcPr127 (SEQ ID NO 12), to which JP62 did not react, differed by onlyone and two nucleotides, respectively, from the sequence of JP62(accession number D13453). Therefore, the chosen hybridization andwashing conditions were very stringent and that even single mismatchesabolish hybridization in this assay.

6. Discrimination Between Subtypes

After careful comparison of all available type 1 coding sequences, twosubtypes (1a and 1b) can clearly be distinguished, with an averagegenome homology of 79%. In the 5′ UR, only 2 mutations were observedbetween HCV-J and HCV-1 in the region of the nested PCR product,resulting in 98.8% homology. Although only 2 mutations were presentbetween HCV-1 (1a) and HCV-J (1b), the A-to-G transition observed atposition −99 occurs in all type 1b isolates studied so far. Therefore,hybridization to probe HcPr138 (SEQ ID NO 7), which spans the region ofthe G substitution, is indicative of a type 1b isolate.

When comparing all available 5′ UR sequences of type 3 (presentinvention; Bukh et al., 1992; Chan et al., 1992; Lee et al., 1992), theisolates could be divided into two groups according to the presence of acommon G (type 3a; HcPr140, SEQ ID NO 15) or a more rare A (type 3b;HcPr139, SEQ ID NO 16) at position −139. Discrimination between types 2aand type 2b (or K2a and K2b) could be made in the variable regions asreported above.

The combination of all these type- and subtype-specific probes for type1 and 3 (Table 4) allowed us to separate the 17 Brazilian sera whichpreviously had been characterized as type 1 into 8 type 1a and 9 type 1bsera. Three of the four type 3 sera formed hybrids with the type 3aline. Different molecules in the cPCR fragment of the co-infected serumBR23 hybridized with the lines for type 1b and type 3a (FIG. 3, strip8).

Another 38 Brazilian sera (BR24 to BR61) were tested in this new LiPA.The most dominant criterium for the selection of these sera was theabsence of antibodies for NS4 and NS5 epitopes, since earlier reportsshowed that there was a low degree of cross-reactivity between type 2and type 3 anti-NS4 antibodies with type 1 NS4 antigens (Chan et al.,1991). Of the 38 Brazilian sera, 12 could be typed as type 1a, 14 astype 1b, 9 as type 3a, 2 as type 3b and a coinfection of type 1b and 3a.It was concluded that all the tested Brazilian sera could be typed. Itremains to be determined whether the discrimination between type 3subtypes is relevant. As no sequence data from the 5′ UR of the Ta andTb isolates (Mori et al., 1992) has been published, our division intotype 3a and 3b is still tentative. More data about the serology and thesequences of the open reading frame are needed to confirm type 3 andtype 4 subtyping.

7. Identification of Type 4 Isolates and Incorporation of Type4-Specific Probes in the LiPA

PCR fragments amplified from 6 Burundian sera (BU74 to BU79) failed toreact with any of the 16-mers on the strips. Three PCR fragments fromthese Burundian samples (BU74: accession number D13449, which wasidentical to BU76, and BU79: accession number D13450; FIG. 2) werecloned and sequenced. Sequences that were clearly different from most ofthe previously described types were obtained. The Burundian samples arerelated to each other, and to Z6 (Bukh et al., 1992) and show higherhomologies to type 1 than to type 3 or type 2. However, most of thedifferences with type 1 were again located in the variable regions. Themost surprising finding was the presence of one extra nucleotide in BU74and BU76 between the positions −139 and −140. These results argue infavor of the existence of new HCV type(s) or subtype(s), which will beprovisionally called type 4. The sequences of the 5′ UR of the virusthat could be amplified from these African sera were strongly divergentfrom the previously described types. Therefore, these isolates have beententatively designated as type 4. Similar sequences communicated in thestudy of Bukh et al. (1992), also originated from Africa, although onewas from Denmark. FIG. 2 shows that in the region between nucleotides−291 and −55, as many as 8 nucleotide variations are possible withinthis group. It is likely that type 4 is further composed of severalsubtypes, or that these subtypes are divergent subtypes of type 1.

After obtaining these data, the LiPA was improved in three ways. First,oligonucleotide HcPr142 (SEQ ID NO 20), carrying one degeneration, waschosen from a highly conserved region as universal HCV probe for theconfirmation of the presence of the PCR product (Table 4). Secondly,three oligonucleotides were synthesized for identification of the type 4sequences (HcPr144, SEQ ID NO 17 with one degeneration, HcPr145, SEQ IDNO 18 and HcPr146, SEQ ID NO 19; Table 4). Thirdly, a universal type 2probe was selected outside the variable regions (HcPr147, SEQ ID NO 8,Table 4), since a universal probe for the detection of type 2 could notbe chosen from the regions between positions −170 and −155 and betweenpositions −132 to −117.

With this version of the LiPA, the 6 PCR fragments from the Burundiansera (Table 3) were classified as type 4 as expected (FIG. 3, strip 6and 7). Two Gabonese sera, 4 sera from the Netherlands and 6 Belgiansera were also included in the screening. From GB80 a type 4 HCV 5′ URcould be amplified, which was cloned and sequenced (FIG. 2). The otherGabonese serum GB81 showed a coinfection of a variant of type 2 (clonedand sequenced, FIG. 2) and type 4. The latter gave the same typingpattern as BU79 (FIG. 3, strip 7). To establish whether reaction of GB81with the type 2 and type 4 probes was caused by unexpectedcross-reactivity between typing probes, or merely the result of acoinfection , the cPCR product was cloned and 17 individual colonieswere subjected to PCR and HCV LiPA. Ten (59%) colonies contained type 4inserts and seven were type 2 (41%), clearly indicating theco-circulation of 2 types of HCV in the same serum. For the threeNE-sera which were negative in the Ortho RIBA test and positive (NE71),indeterminate (NE72) or negative (NE73) in the Inno-LIA HCV Ab test, itcould be shown that type 3a isolates were present. The fourth NE serum,which showed good reactivities in both Ortho RIBA and INNO-LIA,contained a type 1a isolate. Finally, from the 6 Belgian sera analyzed,BE64 to BE67 were infected with type 1b strains. One patient of Italianorigin (BE68) had a type 2a infection, and BE69 contained type 3asequences. The latter was obtained from a case of chronic, viral-likeNANB hepatitis, but was negative in all second generation assays andanti-NS3, anti-E1, and anti-E2 research assays. This serum had a verylow virus titer and became weakly positive only after the second roundof PCR in four different samples taken during 2 years, showing the needfor nested cPCR in HCV diagnosis. The sequence of the nested PCRfragment was identical with BR56. This was not surprising, since type 3strains show very little sequence variation.

In total, 19 different oligonucleotides were used for this version ofthe LiPA strips as shown in FIG. 3. Because some of the oligonucleotidesare directed against the same HCV subtype, probe HcPr156 (SEQ ID NO 22)was pooled with HcPr158 (SEQ ID NO 24) for type 2a. The oligonucleotidesagainst type 4 were applied separately because too little sequenceinformation from the coding region is known at this moment and hence, nodivision into subtypes (if any) can be made as yet. The presence of anextra base in some of the type 4 sequences can form the basis forfurther attempts to subtype this group. The results obtained with somerepresentative sera are shown in FIG. 3. The interpretation of thesestrips is given in Table 2.

In this study, 61 PCR-positive Brazilian HCV sera were typed. Twenty(33%) sera had a type 1a HCV infection, 23 (38%) were type 1b, one(1.5%) type 2a, 15 (24.5%) type 3, and two (3%) sera with coinfectionswere found. The recognition of coinfected sera is illustrated by BR23(FIG. 1, lane 9; FIG. 3, strip 8). The remaining 20 sera were collectedfrom 5 different countries; 8 of the sera originated from two Africancountries.

In a minority of the cases such as was the case for BE67, a type 1b PCRfragment recognized the 3b subtype probe HcPr139 (SEQ ID NO 16). Thiscan be explained by assuming that the 1b sequence of serum BE67 has an Ainstead of a T at position −139. The results obtained with the JP62(accession number D 13453) sequence, where one mismatch in theoligonucleotide abolishes the hybridization signal, further supportsthis assumption. Since isolate-specific mutations are scatteredthroughout the 5′ UR, it is possible that an isolate of a given typealso hybridizes to a subtyping probe of another type (see FIG. 3, strips6 and 7). Such reactivities merely indicate the presence of the sequenceof the subtyping probe in the isolate studied. However, reactivitieswith multiple typing probes were never observed, unless a serum wascoinfected, as investigated for GB81.

In general, when a type 1a cPCR product hybridized on the LiPA, thesequence of the probes HcPr124 (SEQ ID NO 5), HcPr125 (SEQ ID NO 6) andHcPr142 (SEQ ID NO 20) must be present in the nested cPCR fragment.Consequently, 48 (26%) by of 184 by (FIG. 2) are immediately known.Following the same reasoning, it can be calculated that for isolatessimilar to the HCV J type 33%, to the HC J6 type 35%, to the BR56 type34%, to the Z6 and BU77 type 26%, to the BU74 type 41% and to the BU79type 32% of the sequence is known. However, it must be taken intoaccount that due to the degeneration of some of the 16-mers, someinformation is lost and, hence, these percentages are maximum scores.Nevertheless, this approach supports the idea of the sequencing by thehybridization principle (Strezoska et al., 1991).

When comparing LiPA with antibody reactivity of these sera in ourInno-LIA HCV Ab assay (Table 3) some correlations between genotypes andtheir phenotypes (serotype) emerge. The type 3 and 4 sera from Belgium,the Netherlands, Gabon, and Burundi all react very weakly positive,indeterminate, or negative in the second generation antibody assays. Theweakly positive reaction is mostly caused by anti-core antibodies,whereas antibodies against the IAA NS4 and NS5 epitopes are usuallyabsent. This is in agreement with the high conservation of coresequences encoding only slightly different epitopes which allowimmunological cross-reaction. Epitopes for the NS4 and NS5 region arelocated in highly variable regions, disabling most of the immunologicalcross-reaction. As the current antibody assays contain type 1 epitopes,it is possible that a few percent of type 2, type 3, and type 4 infectedsera will show a negative result. However, the conclusion of lack ofcross-reaction of the type 3 Brazilian sera with type 1 NS4 and NS5antigens cannot be drawn from our results (Table 3). For the 14 randomlychosen sera (BR24 to BR37; Table 3), there was 100% correlation betweenthe LIA reactivity and the 9 type 1 viruses. From four type 3 sera, two(BR34 and BR36) reacted with NS4 and three (BR33, BR34 and BR35) withNS5. BR37 was not taken into account because of the coinfection. Whenall serological data of the 77 sera infected by a single type wereanalyzed, 58% and 44% of the type 1 sera recognized the NS4, and NS5epitopes, respectively. These percentages are rather low and due to theselection criteria. For the type 3 sera, 37% and 53% were reactive withthe NS4 and NS5 epitopes, respectively. It is possible that highercross-reactivities are observed in high-risk groups, such as in thosesamples obtained from Brazil, as compared with results in European blooddonors (present invention and Chan et al., 1992). Such cross-reactingsera could be induced by multiple infections, some of which occursimultaneously, but others might occur after one another. A previousanti-HCV memory could be boosted by new HCV infections and result inco-circulation of viruses of one type with antibodies mainly directedagainst another type. Such an explanation is plausible for serum BR56,which has been typed as HCV type 3, but contained antibodies to type 1core, E1, E2, NS3, NS4, and NS5 (data not shown). It remains to bedetermined whether anti-type 3 antibodies are present in this serum.

Besides the differences in immune response, different HCV types couldalso show different progression to long-term liver disease, as hasalready been reported (Okamoto et al., 1992a).

In conclusion, the LiPA allows a rapid determination of the type of HCVinfection. This assay has the ability to discriminate between 4different HCV types and 8 subtypes, and is a good means for determiningnew types.

Moreover, this assay can be further improved by, for example, replacingthe cPCR reactions by the RNA-capture PCR. Finally, this assay couldprove to be instrumental in further establishing the relation betweengenotypes, future serotypes, and the clinical status or outcome of thedisease.

8. Identification of New Types and Subtypes and Probes Useful for theirClassification.

Isolates BE82, BE90, BE91, BE92, BE93, BE94, BE95, BE96, BE97, BE98,obtained from Belgium; GB48, GB116, GB358, GB569, GB549, GB809, GB487,GB724, and GB438, obtained from Gabon; CAM600 and CAM736, obtained fromCameroun; were retained for further study because aberrant reactivitieswere observed after genotyping by means of a LiPA including probes 5 to27 according to examples 3 and 4. The sequences of the 5′ untranslatedregion were obtained after nested PCR by means of primers with SEQ IDNO. 1, 2, 3 and 4, cloning, and sequencing as described in example 2.Sequence information was obtained in the NS5 coding region for most ofthese isolates, and an alignment with known sequences is presented inFIG. 5. The homologies of NS5 nucleic acid and amino acid sequences ofrepresentative isolates for each subtype with the sequences of publishedisolates is presented in Table 6. This calculation allows classificationinto types and subtypes, as presented in FIG. 4. Nucleotide sequencealignment of the 5′ untranslated regions of these new isolates with someprototype sequences is also presented in FIG. 4. Several mutations canbe observed compared to the HCV-1 sequence. As identical mutations inthe 5′ untranslated region correlate with similar sequences in thecoding region, such mutations are employed in the present invention todesign new type and subtype-specific probes.

BE82, a subtype 1b isolate, showed a C mutation at position −94, andtherefore could not react with probe 7. After sequencing of the NS5region, it could be concluded that this isolate belonged to subtype 1b.Therefore, probe 30, including a degeneration of T and C at position−94, should enable better genotyping of subtype 1b.

BE90, another subtype 1b isolate, showed a T mutation at position −159and a G mutation at position −126, and therefore only reacted with theuniversal probes 20 and 27 and the subtyping probe 7. Sequencing of theNS5 region taught that the isolate belonged to subtype 1b. Probe 28,including a degeneration of T and C at position −126 should enablebetter genotyping of types 1 and 6.

Isolate BE92 reacted only with probes 8 and 26 in addition to theuniversal probes 20 and 27. Thus, this isolate could be classified astype 2, but could not be subtyped because no reactivity with probes 23,24, 25, or 26 could be observed. After sequencing, two new motifs couldindeed be observed: GGACCCAGTCTTCCTG, covered by probe 33, andTGCCTGGTCATTTGGG, covered by probe 34. Sequencing of the NS5 regionindeed revealed homologies with type 2a and 2b isolates compatible withclassification within the same type, but in another subtype which is theproposed subtype 2c.

Isolates BE93 and BE94 did not show any reactivity with the subtypingprobe 14. After sequencing the 5′ untranslated region and the NS5region, it was concluded that these isolates belonged to the 3a subtype.Therefore, a probe containing a C and A degeneration at position −118like probe 35, should allow better genotyping of subtype 3a.

Isolates GB48, GB116, GB358, and GB569 showed positive hybridizationsignals on probe 17 and 19 in LiPA, indicating similarity to thepreviously reported type 4 isolates, but isolates GB549 and GB809 onlyreacted with the universal probes. The sequences of parts of the 5′untranslated region and NS5 were obtained. From FIGS. 5 and 6 and Table6, it can be concluded that the isolates represented by GB358 belong tothe same subtype of type 4, which is the proposed subtype 4a. However,both GB549 and GB809 show lower homologies to the subtype 4a, 4b and 4disolates, and also to each other, but GB809 seems to belong to the samesubtype as Z4. These homologies are compatible with classification intothe same type 4, but into a different subtype of type 4: subtype 4e forGB549 and subtype 4c for GB809 are proposed. Sequences obtained fromisolates GB116, GB358, and GB569 all showed the motifs AATCGCCGGGATGACC,detectable with probe 38 and TTTCCGGGCATTGAGC, detectable with probe 19.Thus, probes 38 and 19 are useful for detection and classification ofsubtype 4a. Probe 38 is specific for subtype 4a, 4b, 4d, 4f and 3b,while probe 19 recognizes subtypes 3b, 4a and 4d, but also hybridizes tothe new types 3c and 4f. Interestingly, the new subtype 3b sequenceHCV-TR should cross-react with these probes. However, 3b can still beclassified as type 3 because of the reactivity with the type 3-specificprobe 21.

GB549 also shows characteristic motifs. Motif AATCGCCGGGACGACC can bedetected by probe 40 and the sequence AATGCCCGGCAATTTG is detectablewith probe 41. Thus, probes 40 and 41 are useful for subtyping ofsubtype 4b.

Reactivities identical to GB809 were obtained on LiPA with two samplesobtained from Cameroun: CAM600 and CAM736. After sequencing the NS5region, it could be concluded that these samples belong to the samesubtype as GB809, and after sequencing the 5′ untranslated region, twoidentical motifs were again detected as those already present in GB809.Thus, it appears that the motif AATCGCCGAGATGACC, detectable with probe42, and AATGCTCGGAAATTTG, detectable with probe 43, are characteristicfor subtype 4c, and that probes 42 and 43 are useful for detection andclassification of subtype 4c. However Z4, which shows homology in the E1region compatible with classification into the same subtype 4c, shows511R sequences which are again unique and may be detected by probes 7,50 and 53.

New sequences were detected in the 5′ untranslated region of isolatesGB487, GB724 and BE97. A new subtype classification, not based onsequence information of the coding region, is tentatively proposed forthese isolates. All three isolates show the sequence TGCCTGGAAATTTGGG,detectable with probe 50. GB487 shows the unique sequenceAATCGCCAGGATGACC, detectable with probe 49, and is tentativelyclassified as subtype 4h. GB724 and BE97 both contain the sequenceGGAATCGCCAGGACGA, detectable with probe 53, and are tentativelyclassified as subtype 4g.

Type 4 isolates usually show a T at position −238 and a A at −235.Therefore, probes 37, 38, and 51 should enable better genotyping of type4.

In another example, BE95, which only hybridized to probes 7 and 17 inthe LiPA shows low homologies in the coding region of about 68% with allother isolates, except BE96 which shows an homology to BE95 compatiblewith classification into the same subtype, which is the proposed subtype5a. BE95, BE96, and SA1 all show the same motifs GAGTGTCGAACAGCCT,detected with probe 44; AATTGCCGGGAYGACC, detectable with probes 45 and47; and TCTCCGGGCATTGAGC, detectable with probe 46. Thus, probes 44, 45,46 and 47 are useful for genotyping of type 5a.

Sequences have been published by Bukh et al. (1992), which contain aunique CA insertion between positions −144 and −145. These isolates aretentatively classified as type 6 and can be detected by means of probe48.

A new type of hepatitis C virus was discovered in isolate BE98, whichonly reacted with probe 19 on LiPA. The sequence of the 5′ untranslatedregion contains the new motif GAATCGCCGGGTTGAC that can be detected bymeans of probe 54. Sequencing of the core region revealed sequencesshowing about equally distant homologies to genotypes 3a and 3b, and anew type 3c is proposed for this prototype sequence.

Isolate GB438 contains sequence motifs which are typical for subtype 4a,detectable with probes 38 and 19, but still shows a different sequencein the E1 region, representing a new subtype within type 4, which wasdesignated subtype 4f. Discrimination from subtype 4a may be performedby means of probes with SEQ ID NO 51 and 7.

Probes 29, derived from the sequence of BE90, and probes 51 and 52,derived from the sequence of GB724, may be useful to improve genotypingof certain HCV types or subtypes.

Example 9 Calculation of Nucleotide Distances

Phylogenetic Analysis.

Previously published sequences were taken from the EMBL database,release 35. Other sequences were analysed by the inventors and have beendeposited in the DDBJ database. Sequences were presented in a sequentialformat to the Phylogeny Inference Package Version 3.5c (Felsenstein,March 1993). Only sequences with identical lengths were included in thesimilarity calculations. The programs employed were DNADIST, PROTDIST,DNAPARS, PROTPARS, NEIGHBOR, SEQBOOT, CONSENSE and DRAWTREE. DNA maximumlikelihood distance matrices were produced by DNADIST using the Kimura2-parameter setting. A bootstrapping analysis was run using SEQBOOT,with 2000 repetitions. All these matrices were further analyzed inNEIGHBOR, using the Neighbor-Joining settings and in CONSENSE tocalculate the consensus tree. The SEQBOOT dataset was also analyzed inthe DNAPARS program on 1130 repetitions. Deduced protein sequences wereanalyzed in PROTDIST followed by NEIGHBOR. Finally, the program DRAWTREEwas used to create a′ graphic output of the phylogenetic tree. Allanalyses were done on a SUN SPARC IPX computer station.

The NS5 Region.

By using the primer set described by Enomoto et al. (1990), weamplified, cloned and sequenced 340 by long NS5b PCR fragments from 13different isolates. The nucleotide sequences were used to create aphylogenetic tree using the DNADIST program of the PHYLIP 3.5c package(Felsentstein, 1993). A diversity of 6 major groups or ‘types’ isevident for this unrooted tree. Each group could be further subdividedinto two ore more subgroups or ‘subtypes’. The following clusters(groups consisting of closely related isolates) were created: 1a, 1b,2b, 3a, 3b, 4a and 5a. This clustering appeared in 100% of the bootstrapresampled data sets using the programs SEQBOOT/DNADIST/NEIGHBOR/CONSENSEon 2000 repetitions. The bootstrapped DNAPARS analysis yielded a similarclustering. From the DNADIST matrix, the molecular evolution distancesbetween isolates, subtypes and types could be calculated. Only the aboveindicated separated clusters were included in these calculations.Between isolates in one subtype, this distance ranged from 0.0148 to0.1064 (mean 0.0623; SD 0.0181). The distance between subtypes rangedfrom 0.1654 to 0.2675 (mean 0.2312; SD 0.0182) and that between typesfrom 0.3581 to 0.6549 (mean 0.4942; SD0.0485). However, some exceptionalcases appeared.

The distance between HC-J6 and isolate BE92 was 0.1539, a low value fordistances between distances, but far above any value obtained betweenisolates belonging to the same subtype. NS5 nucleotide sequence homologybetween HC-J6 and BE92 was 86.2%. The bootstrapped DNA datasetsclustered both sequences in 98.8% of the cases, which is anargumentation for a subtype 2a classification, but the molecularevolutionary distance and the sequence of the 5′ UR of BE92 allowed usto tentatively classify this isolate as subtype 2c. GB809 could bepositioned at a mean distance of 0.1509 (min./max.=0.1384/0.1597) fromthe type 4a cluster. A maximum homology of 87.4% exists between GB809and GB358 (type 4a). However, these data, together with the observedvariations in the 5′UR allowed us to create a new type 4 subtype, 4c.GB549 represents the new subtype 4e and has distances of 0.2426 fromcluster 4a; 0.2403 from subtype 4c and 0.1738 from GB438 at thenucleotide level. Isolate GB438 possibly represents another new type 4subtype, tentatively designated 4f.

The Core/E1 Region.

Calculation of the phylogenetic tree for the core/E1 region betweennucleotides 378 and 957 using the DNASDIST program resulted in therecognition of six major branches, representing the 6 differentgenotypes. The following clusters could be delineated with a 100%certainty from the bootstrap resampling analysis on 2000 repetitions:subtype 1a, 1b, 2a, 2b, 3a and 4a. The clustering is irrelevant forsubtype 4c, 4e, 4f and 5a because they are only represented by oneisolate. Based on the DNADIST matrix, the molecular evolutionarydistance for isolates belonging to the same subtype ranged from 0.0402to 0.111 (mean 0.0772, SD 0.0197), between subtypes from 0.1864 to0.3535 (mean 0.2833, SD 0.0350) and between types from 0.3824 to 0.6230(mean 0.4894, SD 0.0554).

The distances from the DNADIST matrix provided further evidence for theexistence of at least 4 different subtypes in type 4. Type 4a has a meanmutual distance of 0.0083; while the mean with type 4c, 4e and 4f was0.2602. Subtype 4c and 4f were separated from 4e by respectively, 0.2047and 0.1864, while the distance between 4c and 4f was 0.2316.

The NS3/4 Region.

DNA sequences containing the previously described type 3a epitope region(Stuyver et al., 1993a) and other sequences of the EMBL databank wereused to calculate the nucleotide distances using DNADIST and NEIGHBOR.From the DNADIST matrix, the molecular evolutionary distances betweenisolates ranged from 0.0407 to 0.1181 (mean 0.0855, SD 0.0190), betweensubtypes from 0.2281 to 0.2603 (mean 0.2416, SD 0.0098) and betweentypes from 0.4052 to 0.6247 (mean 0.4889, SD 0.0531).

Example 10

As described in the introduction to the examples and in previousexamples, variable regions in the 5′UR are expected to containgenotype-specific sequences also in newly discovered genotypes, asexamplified in example 8, and consequently, such new genotype-specificmotifs should again be detectable by means of the genotype-specificprobes as described in example 8. Therefore, probes 32, and as describedin example 8, probes 31, 33, 34, 37, 38, 44, 45, and 46 were synthesizedand applied to nitrocellulose membranes and line probe assays withbiotin-labelled PCR fragments was performed as described in example 3and 4, except for the labelling of the PCR product with biotin which wasnot incorporated from bio-11-dUTP, but from of 5′-biotinylated primerswith SEQ ID NO 3 and 4 or 5′-biotinylated primers with SEQ ID NO 1 and2, during the synthesis of the PCR fragment.

FIG. 6 shows the type-specific hybridization of HCV type 2, but not type1, 5′UR fragments to the probe with SEQ ID NO 32. Both subtype 2a and 2bisolates hybridized specifically to probe 32.

In FIG. 7, the probes with SEQ ID NO 33 and 34 could be shown tohybridize specifically to the genotype 2c PCR product derived from serumBE92, while genotype 2a and 2b sera did not react to these probesalthough a specific hybridization with the respective 2a and 2bgenotype-specific probes could be observed. It is to be understood thatthe new genotype 2c may differ from other genotype 2c subtypesdiscovered recently, and therefore, the alternative names may beproposed for denomination of this subtype.

FIG. 8 shows line probe assays performed with type 4 sera to showspecific hybridization of the most common type 4 sera to probes 37 and38.

FIG. 9 depicts specific hybridization of type 5a sera to probes 44, 45,46, and probe 7, while reactivity of type 4 sera is usually confined toprobe 31 and absent on probes 44 to 46. Therefore, the promiscuity ofthe probe with SEQ ID NO 18 for both type 4 and type 5 isolates, can nowconveniently be overcome by employing, in addition to the probe with SEQID NO 19, probes with SEQ ID NO 37, 38, 44, 45, 46, 7, 30, and 31 fordiscrimination of genotypes 4 and 5.

Example 11

It may be preferable to use other hybridization conditions (temperature,buffers) as those outlined in examples 3 and 4. Therefore, probes withSEQ ID NO 93, 94, 95, and 96 were applied to nitrocellulose membranes.FIG. 10 shows line probe assays with the type 4a serum GB116 and thetype 5a serum BE95, as described in example 4, except for the following:After denaturation of the PCR fragment in NaOH/SDS, 1.0 ml hybridizationsolution (prewarmed at 50° C.) consisting of 3×SSC (Maniatis et al.,(1982) and 1% sodium dodecyl sulphate (SDS), was added to the denaturedPCR product and hybridization was performed in a shaking water bath at50° C. for 2 hours. The strips were washed with the same hybridizationsolution at 50° C. for 30 min, after which the strips were washed andcolor development was performed as described in example 4. In FIG. 10, aclear type-specific reaction can be observed. Therefore, type-specifichybridization has been obtained in other hybridization conditions asdescribed in examples 4 and 10 after having introduced minormodifications to the hybridization probes. For example, the position ofthe probes can be changed in order to achieve more specifichybridization in a certain hybridization condition, for example, theprobe can be positioned in such a way that the type-specific nucleotidesare located in the middle part of the probe. For certain probes toretain specificity in other hybridization conditions, it may also bepreferable to elongate or shorten the contiguous HCV sequence and/or toreverse the sense of the probes to allow genotype-specific hybridizationat a certain preferred temperature or salt concentration. However, insome cases, it may be preferable to include inosines or mismatchingnucleotides to allow genotype-specific hybridization at a certainpreferred temperature or salt concentration. For example, the probe withSEQ ID NO 37, which was able to discriminate between type 4 and 5isolates in tertramethylammoniumchloride buffer as described in example10, was now changed into probe with SEQ ID NO 93(5′-GAGTGTTGTACAGCCTCC-3′) by elongation of the contiguous HCV sequenceat the 3′ end with 2 nucleotides, and probe 93 showed a specificreactivity in SSC/SDS hybridization buffer (FIG. 10). The probe with SEQID NO 44, which was able to discriminate between type 4 and 5 isolatesin tertramethylammoniumchloride buffer as described in example 10, wasnow changed into probe with SEQ ID NO 96 (5′-GAGTGTCGAACAGCCTC-3′) byelongation of the contiguous HCV sequence at the 3′ end with 1nucleotide, and probe 96 showed a specific reactivity in SSC/SDShybridization buffer (FIG. 10). The antisense probe with SEQ ID NO 46,which targets positions −132 to −117 was able to discriminate betweentype 4 and 5 isolates in tertramethylammoniumchloride buffer asdescribed in example 10, was now changed into probe with SEQ IS NO 95(5′-TGCCCGGAGATTTGGG-3′), a sense probe which targets positions −126 to−111, and probe 95 showed a specific reactivity in SSC/SDS hybridizationbuffer (FIG. 10).

This example illustrates the numerous possibilities of developing probesto those skilled in the art for targeting the genotype-specificmutations as presented in FIG. 4, or for targeting the genotype-specificmutations that are present in other new or still to be discoveredgenotypes.

TABLE 1 1a 1b 2a 2b 2c 3a 3b 3c 4a Cha et al. I I II III — IV — — — Nakao et al. Pt K1 K2a K2b — K3 — — — Chan et al. 1 1 2 2 — 3 — — 4 Mori etal. I II III IV — V VI — — Oka moto et al. I II III IV — V VI — — Prototype isolate HCV-1 HCV-J HC-J6 HC-J8 BE92 BR56 HCV-TR BE98 GB358Isolates of the — BE90 — BE91 BE92 BR56 — BE98 GB48 present inventionBE82 BE93 GB116 BE94 GB569 GB215 4b 4c 4d 4e 4f 4g* 4h* 5a 6a 6? Cha etal. — — — — — — V — Naka o et al. — — — — — — — — Chan et al. — — — — —— 5 6 Mori et al. — — — — — — — — Oka moto et al. — — — — — — — — Prototype isolate Z1 GB809 DK13 GB549 GB438 BE97 GB487 SA1 HK1 HK2 Z4Isolates of the — GB809 — GB549 GB438 GB724 GB487 BE95 — HK2 presentinvention CAM600 BE97 BE96 CAM736

TABLE 2 Interpretation of the results shown in FIG. 3 Type Probe BR5BR12 BR18 BR22 BR19 BE95 BU79 BR23 JP63 1 5 + + − − − − − + + 1 6 + + −− − − − + + 1b 7 − + − − − + + + + 2 8 − − + − − − − − + 2 26 − − + − −− − − + 2a 22/24 − − + − − − − − + 2a 10 − − + − − − − − + 3 13 − −− + + − − + − 3 14 − − − + + − − + − 3 21 − − − + + − − + − 3a 15 − −− + − − − + + 3b 16 − − − − + − − − − 4 17 − − − − − + − − − 4 19 − − −− − + + − − 4 18 − − − − − − − − − Universal 155 + + + + + + + + +27 + + + + + + + + + 20 + + + + + + + + + Conjugate + + + + + + + + +Control RESULT 1a 1b 2a 3a 3b 4 4 1b + 3a 1b + 2a

TABLE 3 Final results of HCV LIPA typing and HCV antibody assays IsolateNS4 NS5 Core LIA EIA type BR1 3 3 3 1 1a BR2 3 1 2 1 1a BR3 0 0 0 0 1aBR4 1 0 0 9 1a BR5 3 3 2 1 1a BR6 3 3 2 1 1a BR7 9 0 1 1 1a BR8 0 9 1 91a BR9 0 0 0 0 1b BR10 0 0 0 0 1b BR11 3 0 3 1 1b BR12 1 1 0 1 1b BR13 10 0 9 1b BR14 1 0 0 9 1b BR15 0 0 1 1 1b BR16 1 0 0 9 1b BR17 1 0 0 9 1bBR18 0 0 3 1 2a BR19 3 3 2 1 3b BR20 9 0 1 9 3a BR21 2 3 2 1 3a BR22 0 00 0 3a BR23 3 3 3 1 1b + 3a BR24 2 2 2 1 6.7 1a BR25 3 2 3 1 6.9 1a BR261 2 3 1 6.4 1a BR27 9 0 2 1 7.1 1b BR28 2 3 2 1 6.9 1b BR29 3 3 9 1 6.11b BR30 3 3 2 1 6.6 1b BR31 1 0 2 1 6.8 1b BR32 2 3 2 1 6.8 1b BR33 0 20 1 1.1 3b BR34 1 3 3 1 6.6 3a BR35 9 1 1 1 5.2 3a BR36 2 0 2 1 6.2 3aBR37 1 0 3 1 6.8 1b + 3a BR38 1 3 0 1 7.1 1a BR39 9 0 1 9 2.9 1a BR40 00 2 1 0.9 1a BR41 0 0 3 1 0.3 1a BR42 0 0 1 9 6.0 1a BR43 0 0 2 1 4.7 1aBR44 0 0 2 1 6.7 1a BR45 0 0 1 1 4.7 1a BR46 0 0 9 9 2.2 1a BR47 3 3 2 10.5 1b BR48 0 1 2 1 0.2 1b BR49 0 1 1 1 5.4 1b BR50 1 0 1 1 7.4 1b BR510 0 3 1 7.4 1b BR52 2 3 2 1 4.7 1b BR53 0 0 1 1 7.9 1b BR54 2 3 2 1 1bBR55 0 9 1 9 1.4 3b BR56 2 1 2 1 2.9 3a BR57 1 3 2 1 1.5 3a BR58 1 1 2 17.3 3a BR59 0 1 0 9 0.4 3a BR60 0 3 1 1 1.6 3a BR61 0 0 3 1 2.6 3a JP621 2 2 1 2a JP63 3 3 2 1 1b + 2a BE64 3 3 3 1 1b BE65 0 0 3 1 1b BE66 1 02 1 1b BE67 3 3 2 1 1b BE68 2 1 3 1 2a BE69 0 0 0 0 3a NE70 3 1 3 1 1aNE71 9 9 1 1 3a NE72 0 0 1 9 3a NE73 0 0 0 0 3a BU74 0 0 1 1 4 BU75 0 01 1 4 BU76 0 0 1 1 4 BU77 0 0 2 1 4 BU78 0 0 1 1 4 BU79 0 1 0 9 4 GB80 00 0 0 0.4 4 GB81 0 0 0 0 3.4 2a + 4

TABLE 4 Nucleotide sequence, position and orientation of the primers andprobes. SEQ ID NO. Type Name Position Polarity Sequence from 5′ to3′ (1) Reference  1 Universal HcPr98 −299 + CCCTGTGAGGAACTWCTGTCTTCACGCKato et al., 1992  2 Universal HcPr29 −1   − GGTGCACGGTCTACGAGACCTOkamoto et al., 1991  3 Universal HcPr92 −264 +TCTAGCCATGGCGTTAGTRYGAGTGT Present invention  4 Universal HcPr96 −29  −CACTCGCAAGCACCCTATCAGGCAGT  5 1 HcPr124 −170 + AATTGCCAGGACGACC Kato etal., 1990  6 1 HcPr125 −117 − TCTCCAGGCATTGAGC Kato et al., 1990  7 1bHcPr138 −103 + CCGCGAGACTGCTAGC Kato et al., 1990  8 2 HcPr147 −83  +TAGCGTTGGGTTGCGA Nakao et al., 1991 26 2 HcPr160 −126 − ATAGAGTGGGTTTATCOkamoto et al., 1991  9 2a HcPr136 −168 + TTRCCGGRAAGACTGG Chan et al.,1992 10 2a HcPr137 −117 − TGRCCGGGCATAGAGT Chan et al., 1992 22 2aHcPr156 −165 + CCGGGAAGACTGGGTC Okamoto et al., 1991 24 2a HcPr158−136 + ACCCACTCTATGCCCG Okamoto et al., 1991 11 2b HcPr126 −168 +TTACCGGGAAGACTGG Nakao et al., 1991 12 2b HcPr127 −117 −TGACCGGACATAGAGT Nakao et al., 1991 23 2b HcPr157 −165 +CCGGAAAGACTGGGTC Okamoto et al., 1992 25 2b HcPr159 −136 +ACCCACTCTATGTCCG Okamoto et al., 1992 13 3 HcPr128 −170 +AATCGCTGGGGTGACC Present invention 14 3 HcPr129 −117 − TTTCTGGGTATTGAGCPresent invention 21 3 HcPr154 −103 + CCGCGAGATCACTAGC Present invention15 3a HcPr140 −146 + TCTTGGAGCAACCCGC Chan et al., 1992 16 3b HcPr139−146 + TCTTGGAACAACCCGC Chan et al., 1992 17 4 HcPr144 −170 +AATYCCGGGATGACC Bukh et al., 1992 18 4 HcPr145 −147 + TTCTTGGAACTAACCCPresent invention 19 4 HcPr146 −117 − TTTCCGGGCATTGAGC Present invention20 Universal HcPr142 −115 + TTGGGCGYGCCCCCGC Kato et al., 1990 27Universal HcPr153 −195 + TCTGCGGAACCGGTGA Kato et al., 1990

TABLE 5 Type Sequence 5′ to 3′ SEQ ID NO 1 AATTGCCAGGACGACC  5 1*/6TCTCCAGGCATTGAGC  6 1/6 AATTGCCAGGAYGACC 28 1a/2 CCCCGCAAGACTGCTA 31 1bGCTCAGTGCCTGGAGA 29 1b/3c/5 CCGCGAGACYGCTAGC 30 2/6 CGTACAGCCTCCAGGC 322a CCGGGAAGACTGGGTC 22 2a ACCCACTCTATGCCCG 24 2b ACCCACTCTATGTCCG 25 2cGGACCCAGTCTTCCTG 33 2c TGCCTGGTCATTTGGG 34 3a* CCGCAAGATCACTAGC 36 3aTKTCTGGGTATTGAGC 35 3c GAATCGCCGGGTTGAC 54 4/5 AATYGCCGGGATGACC 174a/4b/4c/4d/4g/4h GAGTGTTGTACAGCCT 37 4e GAGTGTTGTGCAGCCT 393b/4a/4b/4d/4f AATCGCCGGGATGACC 38 3b/4a/4d/3c/4f TTTCCGGGCATTGAGC 19 4eAATCGCCGGGACGACC 40 4e AATGCCCGGCAATTTG 41 4c* AATCGCCGAGATGACC 42 4cAATGCTCGGAAATTTG 43 4h AATCGCCAGGATGACC 49 4h/4g TGCCTGGAAATTTGGG 50 4gGGAATCGCCAGGACGA 53 4f/4e AGTYCACCGGAATCGC 52 4f/4g/6/2a/2b/2cGAGTGTCGTACAGCCT 51 5a/5 GAGTGTCGAACAGCCT 44 5a* AATTGCCGGGATGACC 455a/5 AATTGCCGGGACGACC 47 5a* TCTCCGGGCATTGAGC 46 6a/6 GGGTCCTTTCCATTGG48

TABLE 6 Homology to the NS5 nucleotide (amino acid) sequence of type 1a1b 2a 2b 2c HCV isolates HCV-1 BE90 HC-J6 BE91 BE92 HCV-1-1 100 (100)81.5 (87.5) 67.4 (71.9) 67.1 (72.6) 65.9 (72.6) HC-J1-2 97.1 (99.1) 80.3(86.7) 66.8 (71.9) 67.4 (72.6) 65.6 (72.6) HCV-H-3 97.1 (98.2) 80.6(85.8) 66.8 (69.9) 66.7 (70.8) 66.8 (70.8) HCV-J-4 80.9 (87.6) 89.7(93.8) 67.6 (74.3) 67.1 (73.5) 67.7 (73.5) HCV-JK1-5 81.8 (85.8) 92.9(96.5) 67.7 (70.8) 69.1 (71.7) 68.2 (69.9) HCV-CHINA-6 79.1 (85.8) 94.1(97.4) 65.3 (70.8) 66.5 (69.9) 65.9 (69.9) HCV-T-7 81.5 (87.6) 94.1(98.2) 67.1 (72.6) 68.2 (71.7) 67.4 (71.7) HC-J4.91-8 80.0 (86.7) 91.2(94.7) 67.4 (72.6) 67.9 (71.7) 67.7 (71.7) HCV-TA-9 82.7 (87.6) 91.5(97.4) 67.9 (71.7) 67.9 (70.8) 67.4 (70.8) HCV-JT-10 82.7 (87.6) 91.5(97.4) 67.9 (71.7) 67.9 (70.8) 67.4 (70.8) HCV-BK-11 82.4 (86.7) 94.4(99.1) 66.5 (71.7) 66.5 (70.8) 67.4 (70.8) BE90 81.5 (87.6) 100 (100)66.2 (72.6) 67.1 (71.7) 66.8 (71.7) HC-J6-16 67.4 (71.7) 66.2 (72.6) 100(100) 80.6 (88.5) 86.2 (94.7) HC-J8-17 64.6 (71.5) 66.5 (72.6) 80.6(88.5) 94.7 (99.1) 79.7 (88.5) BE91 67.1 (72.6) 67.1 (71.7) 80.6 (88.5)100 (100) 80.0 (88.5) BE92 65.9 (72.6) 66.8 (71.7) 86.2 (94.7) 80.0(88.5) 100 (100) T1-12 66.5 (71.7) 63.8 (70.8) 62.7 (70.8) 65.0 (71.7)60.9 (69.0) T7-13 66.5 (71.7) 64.4 (70.8) 62.4 (69.0) 64.7 (70.8) 62.7(68.1) BE93 65.0 (70.8) 62.7 (69.9) 62.4 (69.9) 65.0 (70.8) 61.2 (68.1)T9-14 68.2 (75.2) 68.8 (73.5) 63.5 (71.7) 64.4 (72.6) 64.4 (70.8) T10-1567.9 (75.2) 68.5 (73.5) 63.8 (71.7) 65.0 (72.6) 64.4 (70.8) GB48 67.4(75.2) 66.2 (76.1) 67.4 (71.7) 67.4 (72.6) 63.8 (71.7) GB116 67.4 (77.0)65.6 (76.1) 66.8 (69.9) 66.5 (70.8) 62.7 (69.9) GB215 66.8 (72.6) 66.2(75.2) 66.5 (67.3) 66.5 (69.9) 62.4 (67.3) GB358 67.7 (76.1) 67.9 (77.0)66.5 (70.8) 67.1 (71.7) 62.9 (70.8) GB549 68.8 (76.1) 63.5 (74.3) 65.9(71.7) 67.7 (74.3) 62.7 (71.7) GB809 68.8 (73.5) 67.1 (74.3) 67.9 (69.9)68.5 (73.4) 65.3 (69.9) BE95 69.1 (75.2) 69.1 (77.0) 68.5 (73.5) 71.5(76.1) 67.9 (73.5) CHR18-18 67.1 (75.2) 68.2 (77.0) 67.4 (71.7) 67.9(74.3) 66.8 (71.7) 3a 3b 4a 4b 4c 5a HCV isolates BE93 T10 GB358 GB549GB809 BE95 HCV-1-1 65.0 (70.8) 67.9 (75.2) 67.7 (76.1) 68.8 (76.1) 68.8(73.5) 69.1 (75.2) HCV-J1-2 66.2 (70.8) 67.7 (75.2) 68.2 (76.1) 69.7(76.1) 69.4 (73.5) 67.9 (76.1) HCV-H-3 65.0 (69.0) 66.2 (73.5) 66.8(74.3) 67.7 (74.3) 67.7 (71.7) 67.9 (73.5) HCV-J-4 65.6 (70.8) 69.1(75.2) 65.6 (77.0) 67.1 (77.0) 65.6 (74.3) 68.8 (77.9) HCV-JK1-5 63.8(69.9) 67.4 (71.7) 64.1 (75.2) 64.7 (74.3) 66.5 (74.3) 68.2 (76.1)HCV-CHINA-6 62.1 (68.1) 67.1 (71.7) 65.9 (75.2) 63.8 (72.6) 65.0 (72.6)67.7 (75.2) HCV-T-7 62.4 (69.9) 68.5 (73.5) 67.1 (77.0) 65.3 (74.3) 66.5(74.3) 69.4 (77.9) HC-J4.91-8 65.3 (71.7) 69.1 (75.2) 65.0 (77.0) 65.9(75.2) 66.2 (74.3) 68.2 (77.0) HCV-TA-9 63.5 (69.9) 67.4 (73.5) 64.4(75.2) 64.7 (73.5) 66.5 (72.6) 68.2 (76.1) HCV-JT-10 63.5 (69.9) 67.4(73.5) 64.4 (75.2) 64.7 (73.5) 66.5 (72.6) 68.2 (76.1) HCV-BK-11 62.7(69.0) 67.1 (72.6) 65.6 (76.1) 63.5 (73.5) 65.3 (73.5) 67.0 (76.1) BE9062.7 (69.9) 68.5 (73.5) 67.9 (77.0) 63.5 (74.3) 67.1 (74.3) 69.1 (77.0)HC-J6-16 62.4 (69.9) 63.8 (71.7) 66.5 (70.8) 65.9 (71.7) 67.9 (69.9)68.5 (73.5) HC-J8-17 63.5 (70.8) 64.7 (72.6) 65.6 (71.7) 65.9 (74.3)67.4 (73.5) 68.2 (76.1) BE91 65.0 (70.8) 65.0 (72.6) 67.1 (71.7) 67.7(74.3) 68.5 (73.5) 71.5 (76.1) BE92 61.2 (68.1) 64.4 (70.8) 62.9 (70.8)62.7 (71.7) 65.3 (69.9) 67.9 (73.5) T1-12 95.6 (98.2) 79.7 (85.8) 70.6(76.1) 68.2 (75.2) 69.1 (74.3) 67.1 (69.9) T7-13 93.8 (95.6) 80.3 (87.6)70.6 (77.9) 67.9 (77.9) 70.3 (76.1) 67.7 (70.8) BE93 100 (100) 78.8(95.8) 70.0 (76.1) 66.2 (75.2) 69.4 (74.3) 67.4 (69.0) T9-14 77.9 (85.0)98.5 (99.1) 72.4 (78.8) 71.2 (82.3) 70.6 (77.9) 68.2 (73.5) T10-15 78.8(85.8) 100 (100) 72.9 (78.7) 72.1 (82.3) 70.6 (77.9) 67.9 (72.6) GB4869.4 (76.1) 72.7 (77.0) 97.1 (98.2) 80.0 (83.2) 86.2 (92.0) 69.1 (69.9)GB116 70.0 (74.3) 72.9 (77.0) 96.2 (98.2) 78.8 (83.2) 85.2 (92.0) 68.8(69.9) GB215 68.5 (74.3) 71.5 (75.2) 93.8 (96.5) 80.0 (83.2) 86.2 (92.0)68.2 (67.3) GB358 70.0 (76.1) 72.9 (78.8) 100 (100) 79.4 (85.0) 87.4(93.8) 69.7 (70.8) GB549 66.2 (75.2) 72.1 (82.3) 79.4 (85.0) 100 (100)79.7 (87.6) 67.1 (70.8) GB809 69.4 (74.3) 70.6 (77.9) 87.4 (93.8) 79.7(87.6) 100 (100) 68.2 (69.9) BE95 67.4 (69.0) 67.9 (72.6) 69.7 (70.8)67.1 (70.8) 68.2 (69.9) 100 (100) CHR18-18 65.3 (67.3) 66.2 (70.8) 66.8(69.0) 63.8 (69.0) 66.5 (69.0) 92.9 (94.7)

REFERENCES

Barany F (1991). Genetic disease detection and DNA amplification usingcloned thermostable ligase. Proc Natl Acad Sci USA 88: 189-193.

Bej A, Mahbubani M, Miller R, Di Cesare J, Haff L, Atlas. R (1990)Mutiplex PCR amplification and immobilized capture probes for detectionof bacterial pathogens and indicators in water. Mol Cell Probes 4:353-365.

Bukh J, Purcell R, Miller R (1992). Sequence analysis of the 5′noncoding region of hepatitis C virus. Proc Natl Acad Sci USA 89:4942-4946.

Bukh J, Purcell R, Miller R (1993) At least 12 genotypes of hepatitis Cvirus predicted by sequence analysis of the putative E1 gene of isolatescollected worldwide. Proc. Natl. Acad. Sci. USA 90: 8234-8238.

Cha T, Beal E, Irvine B, Kolberg J, Chien D, Kuo G, Urdea M (1992) Atleast five related, but distinct, hepatitis C viral genotypes exist.Proc Natl Acad Sci USA 89: 7144-7148.

Chan S, Simmonds P, McOmish F, Yap P, Mitchell R, Dow B, Follett E(1991) Serological responses to infection with three different types ofhepatitis C virus. Lancet 338: 1991.

Chan S, McOmish F, Holmes E, Dow B, Peutherer J, Follett E, Yap P,Simmonds P (1992a) Analysis of a new hepatitis C virus type and itsphylogenetic relationship to existing variants. J Gen Virol 73:1131-1141.

Chan S, Holmes E, McOmish F, Follett E, Yap P, Simmonds P (1992b)Phylogenetic analysis of a new, highly divergent HCV type (type 3):effect of sequence variability on serological responses to infection.In: Hepatitis C virus and related viruses, Molecular Virology andpathogenesis. First Annual Meeting, Venice, Italy. Abstract book D5, 73.

Chomczynski P, Sacchi N (1987) Single step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem162: 156-159.

Choo Q, Richman K, Han J, Berger K, Lee C, Dong C, Gallegos C, Coit D,Medina-Selby A, Barr P, Weiner A, Bradley D, Kuo G, Houghton M (1991)Genetic organization and diversity of the hepatitis C virus. Proc NatlAcad Sci USA 88: 2451-2455.

Compton J (1991). Nucleic acid sequence-based amplification. Nature,350: 91-92.

Duck P (1990). Probe amplifier system based on chimeric cyclingoligonucleotides. Biotechniques 9, 142-147.

Enomoto. N, Takada A, Nakao T, Date T (1990) There are two types ofhepatitis C virus in Japan. Biochem Biophys Res Comm 170: 1021-1025.

Guatelli J, Whitfield K, Kwoh D, Barringer K, Richman D, Gengeras T(1990) Isothermal, in vitro amplification of nucleic acids by amultienzyme reaction modeled after retroviral replication. Proc NatlAcad Sci USA 87: 1874-1878.

Inchaupse G, Abe K, Zebedee S, Nasoff M, Prince A (1991) Use ofconserved sequences from hepatitis C virus for detection of viral RNA ininfected sera by polymerase chain reaction. Hepatology, 14: 595-600.

Jacobs K, Rudersdorf R, Neill S, Dougherty J, Brown E, Fritsch E (1988)The thermal stability of oligonucleotide duplexes is sequenceindependent in tedraalkylammonium salt solutions: application toidentifying recombinant DNA clones. Nucl Acids Res 16: 4637-4650.

Kanai K, Kako M, Okamoto H (1992) HCV genotypes in chronic hepatitis Cand response to interferon. Lancet 339: 1543

Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T,Shimotohno K (1990) Molecular cloning of the human hepatitis C virusgenome from Japanese patients with non-A, non-B hepatitis. Proc NatlAcad Sci USA 87: 9524-9528.

Kubo Y, Takeuchi K, Boonmar S, Katayama T, Choo Q, Kuo G, Weiner A,Bradley D, Houghton M, Saito I, Miyamura T (1989) A cDNA fragment ofhepatitis C virus isolated from an implicated donor of post-transfusionnon-A, non-B hepatitis in Japan. Nucl Acids Res 17: 10368-10372.

Kwoh D, Davis G, Whitfield K, Chappelle H, Dimichele L, Gingeras T(1989). Transcription-based amplification system and detection ofamplified human immunodeficiency virus type 1 with a bead-based sandwichhybridization format. Proc Natl Acad Sci USA, 86: 1173-1177.

Landegren U, Kaiser R, Sanders J, Hood L (1988). A ligase-mediated genedetection technique. Science 241: 1077-1080.

Lee C, Cheng C, Wang J, Lumeng L (1992) Identification of hepatitis Cviruses with a nonconserved sequence of the 5′ untranslated region. JClin Microbiol 30: 1602-1604.

Lizardi P, Guerra C, Lomeli H, Tussie-Luna I, Kramer F (1988)Exponential amplification of recombinant RNA hybridization probes.Bio/Technology 6: 1197-1202.

Lomeli H, Tyagi S, Printchard C, Lisardi P, Kramer F (1989) Quantitativeassays based on the use of replicatable hybridization probes. Clin Chem35: 1826-1831.

Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: a laboratorymanual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Mori S, Kato N, Yagyu A, Tanaka T, Ikeda Y, Petchclai B, Chiewsilp P,Kurimura T, Shimotohno K (1992) A new type of hepatitis C virus inpatients in Thailand. Biochem Biophys Res Comm 183: 334-342.

Nakao T, Enomoto N, Takada N, Takada A, Date T (1991) Typing ofhepatitis C virus genomes by restriction length polymorphism. J GenVirol 72: 2105-2112.

Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H,Tsuda F, Miyakawa Y, Mayumi M (1990) The 5′ terminal sequence of thehepatitis C virus genome. Japan J Exp Med 60: 167-177.

Okamoto H, Okada S, Sugiyama Y, Kurai K, Iizuka H, Machida A, MiyakawaY, Mayumi M (1991) Nucleotide sequence of the genomic RNA of hepatitis Cvirus isolated from a human carrier: comparison with reported isolatesfor conserved and divergent regions. J Gen Virol 72: 2697-2704.

Okamoto H, Sugivama Y, Okada S, Kurai K, Akahane Y, Sugai Y, Tanaka T,Sato K, Tsuda F, Miyakawa Y, Mayumi M (1992a) Typing hepatitis C virusby polymerase chain reaction with type-specific primers: application toclinical surveys and tracing infectious sources. J Gen Virol 73:673-679.

Okamoto H, Kurai K, Okada S, Yamamoto K, Lizuka H, Tanaka T, Fukuda S,Tsuda F, Mishiro S (1992b) Full-length sequences of a hepatitis C virusgenome having poor homology to reported isolates: comparative study offour distinct genotypes. Virology 188: 331-341.

Pozatto G, Moretti M, Franzin F, Croce L, Tiribelli C, Masayu T, KanekoS, Unoura M, Kobayashi K (1991) Severity of liver disease with differenthepatitis C viral clones. Lancet 338: 509

Saiki r, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G, Mullis K,Erlich H (1988). Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase. Science 239: 487-491.

Strezoska Z, Paunesku T, Radosavljevic D, Labat I, Drmanac R,Crkvenjakov R (1991) DNA sequencing by hybridization: 100 bases read bya non-gel-based method. Proc Natl Acad Sci USA 88: 10089-10093.

Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Onishi E,Andoh T, Yoshida I, Okayama H (1991) Structure and organization of thehepatitis C virus genome isolated from human carriers. J Virol 65:1105-1113.

Walker G, Little M, Nadeau J, Shank D (1992). Isothermal in vitroamplification of DNA by a restriction enzyme/DNA polymerase system. ProcNatl Acad Sci USA 89: 392-396.

Wu D, Wallace B (1989). The ligation amplification reaction(LAR)-amplification of specific DNA sequences using sequential rounds oftemplate-dependent ligation. Genomics 4: 560-569.

Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M,Higashi Y, Shibata M, Morishima T (1992) Detection of hepatitis C virusby polymerase chain reaction and response to interferon-therapy:relationship to genotypes of hepatitis C virus. Hepatology 16: 293-299.

1. An isolated polynucleic acid consisting of 15-50 nucleotides capableof specifically hybridizing with an HCV NS5B sequence portion of atarget sequence, said HCV NS5B sequence portion being a nucleic acidsequence encoding a NS5B amino acid sequence defined by any one of SEQID NO:82 to SEQ ID NO:92, or a complement of a nucleic acid sequenceencoding a NS5B amino acid sequence defined by any one of SEQ ID NO:82to SEQ ID NO:92.
 2. The isolated polynucleic acid of claim 1 hybridizingunder conditions allowing detection of homologous target sequencescomprising one or few mismatches.
 3. The isolated polynucleic acid ofclaim 1 hybridizing under conditions allowing detection of completelyhomologous target sequences.
 4. A capture probe or labeled probeconsisting of a binding molecule or a detectable label, and a nucleicacid sequence of 15-50 nucleotides capable of specifically hybridizingwith an HCV NS5B sequence portion of a target sequence, said HCV NS5Bsequence portion being a nucleic acid sequence encoding a NS5B aminoacid sequence defined by any one of SEQ ID NO:82 to SEQ ID NO:92, or acomplement of a nucleic acid sequence encoding a NS5B amino acidsequence defined by any one of SEQ ID NO:82 to SEQ ID NO:92.
 5. Anisolated HCV virus characterized by any of the HCV NS5 nucleotidesequences selected from the group consisting of a nucleic acid sequenceencoding any of SEQ ID NO:82 to SEQ ID NO:92.
 6. A kit for detecting ortyping HCV isolates comprising at least one isolated polynucleic acid ofclaim
 1. 7. A solid substrate comprising the capture probe or labeledprobe of claim 4 immobilized thereon.
 8. An isolated polynucleic acidsequence comprising a polynucleic acid sequence encoding any of aminoacid sequences defined by SEQ ID NO:82 to SEQ ID NO:92, or a complementthereof.
 9. An isolated polynucleic acid comprising 15-50 nucleotidescapable of specifically hybridizing with an HCV NS5B sequence portion ofa target sequence, said HCV NS5B sequence portion being a nucleic acidsequence encoding a NS5B amino acid sequence defined by any one of SEQID NO:82 to SEQ ID NO:92, or a complement of a nucleic acid sequenceencoding a NS5B amino acid sequence defined by any one of SEQ ID NO:82to SEQ ID NO:92.